Distribution of follicles in canine ovarian tissues and xenotransplantation of cryopreserved ovarian tissues with even distribution of follicles.

Ovarian follicles are not homogeneously distributed within the ovarian cortex in several species of mammals. Yet to maximize the reproducibility of experimental results of ovarian transplantation, it is essential to assess the degree of density and distribution of follicles in ovarian tissues before their transplantation. In this study, the ovarian cortex from 13 immature bitches (ten purebred and three mongrels) was sectioned into 1.0- to 1.5-mm3 cubes, those were fixed, sectioned and stained with haematoxylin and eosin. To evaluate the density and distribution of follicles, the mean number of all stages of follicles per square millimetre was calculated after observation under a microscope. The distribution of follicles was considered even when the variance value was lower than 10 or between 10 and 16, with an absolute value of distortion inferior to 1. The mean number of follicles ranged from 3.24 to 28.34/mm2 in 25 ovaries from 13 bitches examined. The variance and distortion ranged from 0.35 to 119.64 and -1.87 to 4.40, respectively. The distribution of follicles within the ovarian cortex was judged uneven in 12 of 25 ovaries. These results indicated that follicles were not homogeneously distributed within the ovarian cortex in a large proportion of ovaries. In addition, cryopreserved ovarian fragments with even distribution of follicles were transplanted to NSG mice with or without 400 U/kg of disialylated erythropoietin (asialo EPO). After removing both sides of ovary, a piece of ovarian fragment was placed under the kidney capsule in both sides of kidney. At 4 weeks after transplantation, the fragments were recovered from the mice and the number of primordial, primary, secondary and antral follicles was counted. Total number of follicles and survival rates of follicles in transplanted fragments with asialo EPO were higher than without asialo EPO in four bitches examined. These findings suggest that asialo EPO might be effective on the follicular survival of canine ovarian tissues after xenotransplantation. Knowing the degree of density and distribution of follicles in ovarian tissues before transplantation is expected to contribute to the precise interpretation of results after transplantation of the ovarian tissues.

Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry.

Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed 'lipoplex', we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis.

Erythropoietin receptor (EpoR) agonism is used to treat a wide range of disease.

The erythropoietin receptor (EpoR) was discovered and described in red blood cells (RBCs), stimulating its proliferation and survival. The target in humans for EpoR agonists drugs appears clear-to treat anemia. However, there is evidence of the pleitropic actions of erythropoietin (Epo). For that reason, rhEpo therapy was suggested as a reliable approach for treating a broad range of pathologies, including heart and cardiovascular diseases, neurodegenerative disorders (Parkinson's and Alzheimer's disease), spinal cord injury, stroke, diabetic retinopathy and rare diseases (Friedreich ataxia). Unfortunately, the side effects of rhEpo are also evident. A new generation of nonhematopoietic EpoR agonists drugs (asialoEpo, Cepo and ARA 290) have been investigated and further developed. These EpoR agonists, without the erythropoietic activity of Epo, while preserving its tissue-protective properties, will provide better outcomes in ongoing clinical trials. Nonhematopoietic EpoR agonists represent safer and more effective surrogates for the treatment of several diseases such as brain and peripheral nerve injury, diabetic complications, renal ischemia, rare diseases, myocardial infarction, chronic heart disease and others.

Subventricular zone-derived oligodendrogenesis in injured neonatal white matter in mice enhanced by a nonerythropoietic erythropoietin derivative.

Perinatal hypoxia-ischemia (HI) frequently causes white-matter injury, leading to severe neurological deficits and mortality, and only limited therapeutic options exist. The white matter of animal models and human patients with HI-induced brain injury contains increased numbers of oligodendrocyte progenitor cells (OPCs). However, the origin and fates of these OPCs and their potential to repair injured white matter remain unclear. Here, using cell-type- and region-specific genetic labeling methods in a mouse HI model, we characterized the Olig2-expressing OPCs. We found that after HI, Olig2+ cells increased in the posterior part of the subventricular zone (pSVZ) and migrated into the injured white matter. However, their oligodendrocytic differentiation efficiency was severely compromised compared with the OPCs in normal tissue, indicating the need for an intervention to promote their differentiation. Erythropoietin (EPO) treatment is a promising candidate, but it has detrimental effects that preclude its clinical use for brain injury. We found that long-term postinjury treatment with a nonerythropoietic derivative of EPO, asialo-erythropoietin, promoted the maturation of pSVZ-derived OPCs and the recovery of neurological function, without affecting hematopoiesis. These results demonstrate the limitation and potential of endogenous OPCs in the pSVZ as a therapeutic target for treating neonatal white-matter injury.

Erythropoietin, but not asialoerythropoietin or carbamyl-erythropoietin, attenuates monocrotaline-induced pulmonary hypertension in rats.

Erythropoietin (EPO) has long been utilized for the treatment of renal anemia. The erythropoietin receptor (EPOR) is also expressed in the cardiovascular and central nervous systems in addition to an erythroid lineage, to provide an organoprotective role against several types of cellular stress. Pulmonary hypertension (PH) is a poor prognostic disease caused by primary and secondary pulmonary vascular injury. We observed the effects of EPO derivatives on monocrotaline-induced PH in rats on the supposition that EPO may protect small arteries from injury. Asialoerythropoietin (AEPO) lacks sialic acids in the termini of carbohydrate chains that results in rapid clearance from blood. Carbamyl-erythropoietin (CEPO) interacts with EPOR/ßc heterodimers, but not with EPOR homodimers expressed in erythroid cells. Monocrotaline-injected rats were treated with continuous intravenous injection of 2500 ng/kg/day of EPO, AEPO, or CEPO for 21 days, and lung histology, cardiac function, and mRNA expression in the lungs were examined. Wall thickening of small arteries in the lungs and PH were improved by administration of EPO, but not by its non-hematopoietic derivatives, AEPO, or CEPO. Erythropoietin administration increased mRNA expression of the anti-apoptotic molecule, Bcl-xL, and maintained expression of the CD31 antigen. We conclude that lungs may express EPOR homoreceptors, but not heteroreceptors. Adequate serum erythropoietin levels may be essential for pulmonary protective effects.

Construction and characterization of an anti-asialoglycoprotein receptor single-chain variable-fragment-targeted melittin.

Antibody-therapeutic agent conjugation to be delivered specifically to tumor cells is required for many target-based therapeutic strategies. In the present study, a recombinant immunotoxin was constructed by which melittin was fused to an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (C1), and targeting ability and cytolytic efficacy of the fusion protein were studied. Our results suggested that the recombinant 29.4 kDa protein C1M was expressed in Escherichia coli as a soluble style. Binding of C1M to the surface of hepatocellular carcinoma (HCC) cells was confirmed by both immunohistochemistry and flow cytometry assays. C1M kept the hemolytic activity of melittin and exhibited cytolytic capacity to HepG2 cells at a concentration of 1.5 µg/mL, under which erythrocytes would not be lysed. The effects were greatly inhibited by coadministration with asialoorosomucoid, a natural ligand for ASGPR. These results suggested that C1M conferred targeting and ASGPR-specific cytotoxicity to HCC cells. This work makes it possible to further investigate its antihepatoma efficacy in vivo.

Purification of a lectin from Arisaema erubescens (Wall.) Schott and its pro-inflammatory effects.

The monocot lectin from the tubers of Arisaema erubescens (Wall.) Schott has been purified by consecutive hydrophobic chromatography and ion exchange chromatography methods. The molecular weight of this A. erubescens lectin (AEL) was determined to be about 12 kDa by high performance liquid chromatography (HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) methods. AEL could agglutinate rabbit erythrocytes. The haemagglutination activity of AEL was only inhibited by asialofetuin, while monosaccharide did not react. Rat paw edema and neutrophil migration models were used to investigate the pro-inflammatory activity of AEL. AEL (100 and 200 µg/paw) could induce significant rat paw edema. In addition, AEL (100, 200 and 300 µg/mL/cavity) could induce significant and dose-dependent neutrophil migration in the rat peritoneal cavities. Besides, AEL at doses ranging from 100 to 300 µg/mL/cavity could significantly increase the concentration of nitric oxide (NO), prostaglandin E(2 )(PGE(2)) and tumor necrosis factor alpha (TNF-α) in peritoneal fluid. As compared with control animals, 75% depletion in the number of resident cells following peritoneal lavage did not reduce the AEL-induced neutrophil migration. However, pre-treatment with 3% thioglycollate which increased the peritoneal macrophage population by 201%, enhanced the neutrophil migration induced by AEL (200 µg/mL/cavity) (p < 0.05). Reduction of peritoneal mast cell population by chronic treatment of rat peritoneal cavities with compound 48/80 (N-methyl-p-methoxyphenethylamine with formaldehyde) did not modify AEL-induced neutrophil migration. The results provided the basis for identifying the toxic components of A. erubescens and AEL could be a new useful tool for pro-inflammatory research.

Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor.

BACKGROUND: Hepatic failure has been treated successfully with clinical extracorporeal perfusions of porcine livers. However, dog-to-pig and pig-to-baboon liver xenotransplant models have resulted in severe bleeding secondary to liver xenograft-induced thrombocytopenia. Kupffer cells (KC) are abundant phagocytic cells in the liver. KC express the CD11b/CD18 receptor, which has been implicated in chilled platelet binding and phagocytosis through interaction with platelet surface proteins and carbohydrates. We sought to identify the role of KC CD18 in liver xenograft-induced thrombocytopenia. METHODS: Primary pig KC were characterized by flow cytometry, immunoblots, and quantitative polymerase chain reaction. Pig KC were used in inhibition assays with fluorescently labeled human platelets. The CD18 receptor was targeted for siRNA knockdown. RESULTS: Domestic and α1,3-galactosyltransferase double knockout porcine KC cultures were approximately 92% positive for CD18 as detected by quantitative polymerase chain reaction and flow cytometry. Use of CD18 blocking antibodies resulted in reduction of human platelet binding and phagocytosis. Additionally, asialofetuin, not fetuin, inhibited platelet phagocytosis suggesting the involvement of an oligosaccharide-binding site. Furthermore, reduced CD18 expression by siRNA resulted in decreased human platelet binding. CONCLUSIONS: Our data suggest that primary pig KC bind and phagocytose human platelets with involvement of CD18. Further understanding and modification of CD18 expression in pigs may result in a liver xenograft with reduced thrombocytopenic effects, which could be used as a bridge to allogeneic liver transplantation.

Effects of asialo-erythropoietin on pain-related behavior and expression of phosphorylated-p38 map kinase and tumor necrosis factor-alpha induced by application of autologous nucleus pulposus on nerve root in rat.

STUDY DESIGN: this study was designed to examine the neuroprotective effects of asialo-erythropoietin (A-EPO) in a rat model of lumbar disc herniation. OBJECTIVE: to investigate the effects of A-EPO on pain-related behavior, the expression of phosphorylated-p38 (p-p38) mitogen activated kinase, and the expression of tumor necrosis factor alpha (TNF-α) induced by nucleus pulposus (NP) application on the nerve root. SUMMARY OF BACKGROUND DATA: erythropoietin (EPO) has neuroprotective effects in a variety of models of central and peripheral nerve injuries. However, EPO is a hematopoietic growth factor and can therefore cause significant side effects such as thicker blood and promotion of blood clotting. A-EPO is a neuroprotective derivative of EPO that is not hematopoietic. METHODS: female Sprague-Dawley rats (n = 149) were used in this study. NP harvested from the tail was applied to the left L5 nerve root and the rats were then divided into four groups: NP + nontreatment group, no further treatment; NP + A-EPO group, 13.4 microg/kg A-EPO; NP + EPO group, 13.4 microg/kg EPO; and NP + vehicle group, received vehicle. The substances were administered subcutaneously 1 day before surgery and daily for 2 weeks. In the sham group of animals, the L5 nerve root was exposed and NP was not applied. Withdrawal thresholds were determined by the von-Frey test 28 days after surgery. The expressions of p-p38 and TNF-α were assessed by immunohistochemical and immunoblotting analysis. Data were analyzed by unpaired Student t test and Dunnett t test (significance level, P < 0.05). RESULTS: in the NP + nontreatment and NP + vehicle groups, withdrawal thresholds were decreased significantly for 28 days compared with the sham group (P < 0.05). In the NP + A-EPO group, the thresholds were significantly increased on day 28, and in the NP + EPO group, the thresholds were significantly increased on days 21 and 28 (P < 0.05) compared with the NP + nontreatment and NP + vehicle groups. The expression of p-p38 in the NP + A-EPO group was significantly lower than that in the NP + vehicle group on day 1 (P < 0.05). The expression of TNF in the NP + A-EPO and NP + EPO groups was significantly lower than that in the NP + vehicle group on days 1 and 7 (P < 0.05). CONCLUSIONS: A-EPO improved pain-related behavior and reduced the expression of p-p38 and TNF-α. The effect of A-EPO may be related to the inhibitory action of p-p38 and TNF-α in the dorsal root ganglion.

Investigating clearance mechanisms for recombinant activated factor VII in a perfused liver model.

Clearance mechanisms for recombinant activated human FVII (rFVIIa; NovoSeven), a heterogeneously glycosylated protein, have yet to be fully elucidated, but may involve the liver. The effects of the gamma-carboxy glutamic acid (Gla) domain and the sialic acid content of the protein on rFVIIa clearance were investigated following intravenous administration of rFVIIa lacking the Gla domain, des(1-44) rFVIIa and asialo-rFVIIa in pharmacokinetic (PK) studies and perfused rat livers. PK parameters for both rFVIIa and des(1-44) rFVIIa had similar biphasic clearance profiles, as well as half-lives ([t(1/2)]=80 and 88 minutes, respectively), while asialo-rFVIIa was cleared quickly (t(1/2)=21 minutes) with a linear clearance profile. Perfused liver studies with all proteins (10 nM) mirrored the trends in profiles observed in the PK study. rFVIIa and des(1-44) rFVIIa were cleared to a similar extent, 41% and 35%, respectively, after 1 h, whereas plasma-derived FVII from humans (which has a higher sialylation content than rFVIIa) was cleared to a lesser extent (21%). Asialo-rFVIIa, on the other hand, was almost totally cleared and when an excess of asialo-orosomucoid was added to the perfusate, its clearance was significantly reduced (by 34%) and also for rFVIIa, albeit to a lesser extent (by 14%). Together these data suggest that carbohydrate receptor(s) (e.g. the asialoglycoprotein receptor, ASGPR) play a role in asialo-rFVIIa and rFVIIa clearance. In vivo and liver clearance data correlated well showing similar trends and indicated that rFVIIa clearance is not affected by the Gla domain, but rather by a subpopulation of N-glycosylated structures on rFVIIa.

Asialoerythropoietin attenuates neuronal cell death in the hippocampal CA1 region after transient forebrain ischemia in a gerbil model.

BACKGROUND AND PURPOSE: Systemic administration of high-dose recombinant human erythropoietin (rhEPO) is known to attenuate ischemic injury. However, high-dose rhEPO might aggravate ischemic lesions by increasing blood viscosity because of its erythropoietic effects. Asialoerythropoietin (asialoEPO), an EPO derivative with an extremely short plasma half-life, has considerably lesser erythropoietic effect than that of naive EPO. We attempted to determine whether asialoEPO exerts the same neuroprotective effect as naive EPO in a gerbil transient forebrain ischemia model. METHODS: Transient occlusion of both the common carotid arteries was performed in 23 adult gerbils. The drugs (asialoEPO or rhEPO, 10 U/g bodyweight) or phosphate-buffered saline (PBS) were injected intraperitoneally at three times (3 hours before, immediately after, and 24 hours after the ischemic insult). Learning and retention tests were performed on days 6 and 7, respectively, and histological analyses were performed on day 7. RESULTS: Animals treated with asialoEPO and rhEPO showed significant neurological improvement compared to the PBS-treated animals. The number of viable neurons in the CA1 field of the rhEPO-treated (103.57 ± 27.90 cells/mm) and asialoEPO-treated (144.99 ± 34.87 cells/mm) animals was higher than that of the PBS-treated animals (19.53 ± 3.79 cells/mm). Terminal dinucleotidyltransferase-mediated UTP end labeling-positive cells were significantly lower in the rhEPO-treated (33.40 ± 8.13 cells/mm) and asialoEPO-treated (29.28 ± 14.91 cells/mm) animals than in the PBS-treated animals (76.67 ± 8.14 cells/mm). AsialoEPO treatment did not have any effect on erythropoiesis. CONCLUSION: Multiple dosing of asialoEPO, like EPO, could protect the hippocampal CA1 neurons from ischemic damage without affecting erythropoiesis.

Synthesis of a family of amphiphilic glycopolymers via controlled ring-opening polymerization of functionalized cyclic carbonates and their application in drug delivery.

Polymers bearing pendant carbohydrates have a variety of biomedical applications especially in the area of targeted drug delivery. Here we report the synthesis of a family of amphiphilic block glycopolymers containing d glucose, d galactose and d mannose via metal-free organocatalyzed ring-opening polymerization of functional cyclic carbonates generating narrowly dispersed products of controlled molecular weight and end-group fidelity, and their application in drug delivery. These glycopolymers self-assemble into micelles having a high density of sugar molecules in the shell, a size less than 100 nm with narrow size distribution even after drug loading, and little cytotoxicity, which are important for drug delivery. Using galactose-containing micelles as an example, we demonstrate their strong targeting ability towards ASGP-R positive HepG2 liver cancer cells in comparison with ASGP-R negative HEK293 cells although the galactose is attached to the carbonate monomer at 6-position. The enhanced uptake of DOX-loaded galactose-containing micelles by HepG2 cells significantly increases cytotoxicity of DOX as compared to HEK293. This new family of amphiphilic block glycopolymers has great potential as carriers for targeted drug delivery.

Does the concomitant intra-arterial injection of asialoerythropoietin and edaravone mitigate ischaemic mucosal damage after acute superior mesenteric artery thromboembolism in a rabbit autologous fibrin clot model?

To increase the survival rate of patients with acute superior mesenteric artery thromboembolism (ASMAT) treated by catheter thrombolysis, we examined the effects of delivering edaravone and asialoerythropoietin, agents with tissue-protective activities, using a rabbit autologous fibrin clot ASMAT model. Japanese white rabbits (n=32) were randomly separated into four equal groups. 45 min after introducing autologous fibrin clot, Group U received urokinase and heparin; Group E received urokinase and heparin plus edaravone; Group A received urokinase and heparin plus asialoerythropoietin; and Group EA received urokinase, heparin and edaravone plus asialoerythropoietin via a catheter. The intestines were removed 6 h later and intestinal mucosal damage was scored using the Park's injury score. Survival time was assessed. Average mucosal injury was 5.78+/-1.52 (Group U), 2.88+/-0.72 (Group E), 1.90+/-1.23 (Group A) and 1.18+/-1.25 (Group EA). The degree of mucosal injury was significantly lower in Group EA than in Groups U and E (p<0.05). Conversely, there was no significant difference between Group A and Group EA, or between Group A and Group E. The survival times were 31.50+/-13.30 h (Group U), 51.00+/-24.74 h (Group E), 48.00+/-16.97 h (Group A) and 82+/-51.07 h (Group EA); the difference among the four groups was not significant. In conclusion, the concomitant administration of asialoerythropoietin and edaravone reduced mucosal membrane injury significantly compared with edaravone alone. However, to improve the survival of ASMAT rabbit models, the delivery of an appropriate dose of asialoerythropoietin is required, together with the development of methods to assess peripheral recanalisation.

Dual roles for hepatic lectin receptors in the clearance of chilled platelets.

Rapid chilling causes glycoprotein-Ib (GPIb) receptors to cluster on blood platelets. Hepatic macrophage beta(2) integrin binding to beta-N-acetylglucosamine (beta-GlcNAc) residues in the clusters leads to rapid clearance of acutely chilled platelets after transfusion. Although capping the beta-GlcNAc moieties by galactosylation prevents clearance of short-term-cooled platelets, this strategy is ineffective after prolonged refrigeration. We report here that prolonged refrigeration increased the density and concentration of exposed galactose residues on platelets such that hepatocytes, through Ashwell-Morell receptor binding, become increasingly involved in platelet removal. Macrophages rapidly removed a large fraction of transfused platelets independent of their storage conditions. With prolonged platelet chilling, hepatocyte-dependent clearance further diminishes platelet recovery and survival after transfusion. Inhibition of chilled platelet clearance by both beta(2) integrin and Ashwell-Morell receptors may afford a potentially simple method for storing platelets in the cold.

Potentiation of proliferation of some but not all human colon carcinoma cell lines by immobilized hepatic asialoglycoprotein receptor 1.

Twenty-three human colorectal carcinoma cell lines were examined for the binding of recombinant hepatic asialoglycoprotein receptor 1 (ASGR1), which is known to be exclusively expressed on hepatic parenchymal cells. The effects of the binding were assessed by adhesion to and proliferation on immobilized recombinant ASGR1. Recombinant ASGR1 bound strongly to six cell lines and moderately to 15 cell lines out of 23 lines tested, as shown by flow cytometric analysis. The first six cell lines (group A) also exhibited strong adherence to immobilized ASGR1, whereas 11 of the 15 cell lines of the second group (group B) showed significant adhesion with smaller enhancement by ASGR1 than the cell lines in group A. With a representative cell line (DLD-1 cells categorized in group B), a significant portion of the adhesion was inhibited by preincubation of ASGR1 with asialofetuin, a competitive inhibitor of the carbohydrate recognition by ASGR1. The growth rates of 13 cell lines (two of group A and 11 of group B) were significantly accelerated when they were cultured on immobilized recombinant ASGR1. The results indicate that ASGR is a potential organ-specific microenvironmental factor for colorectal carcinoma growth and metastasis formation in livers.

Beneficial effect of desialylated erythropoietin administration on the frozen-thawed canine ovarian xenotransplantation.

PURPOSE: The main drawback of ovarian cryopreservation followed by transplantation is that a large proportion of follicles are lost after transplantation. Thus, effects of erythropoietin (EPO) and desialylated EPO administration on the frozen-thawed canine ovarian xenotransplantation were examined. METHODS: The protective and survival-promoting effects of EPO and desialylated EPO on the follicles of frozen-thawed canine ovaries after transplantation were examined using NOD-SCID mice. Frozen-thawed dog ovarian tissue with 400 U/kg of EPO or asialo EPO was placed into the ovarian bursa. RESULTS: At 4 weeks after the transplantation, the ovaries were removed and subjected to histological examination. The survival rate of early primary follicles was 15.2% in the EPO group and 157.6% in the asialo EPO group, in contrast to 10.1% in the untreated group. CONCLUSIONS: These results demonstrate that administration of asialo EPO could be effectively used to enhance the survival of the follicles of transplanted cryopreserved ovaries.

Isolation, purification, characterization and glycan-binding profile of a d-galactoside specific lectin from the marine sponge, Halichondria okadai.

A lectin recognizing both Galbeta1-3GlcNAc and Galbeta1-4GlcNAc was purified from the demosponge Halichondria okadai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 30 kDa by SDS-PAGE under reducing and non-reducing conditions and 60 kDa by gel permeation chromatography. The pI value of the lectin was 6.7. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the presence and absence of divalent cations. The hemagglutinating activity by the lectin was inhibited by d-galactose, methyl-d-galactopyranoside, N-acetyl-d-galactosamine, methyl-N-acetyl-d-galactosaminide, lactose, melibiose, and asialofetuin. The K(d) of the lectin against p-nitrophenyl-beta-lactoside was determined to be 2.76x10(-5) M and its glycan-binding profile given by frontal affinity chromatography was shown to be similar to many other known galectins. Partial primary structure analysis of 7 peptides by cleavage with lysyl endopeptidase indicated that one of the peptides showed significant similarity with galectin purified from the sponge Geodia cydonium.

Differential induction of immunoregulatory circuits of phagocytic cells by Gal/Gal NAc lectin from pathogenic and nonpathogenic Entamoeba.

INTRODUCTION: Amoebic liver abscess (ALA) is an acute inflammatory disease caused by Entamoeba histolytica. MATERIALS AND METHODS: The key player in the pathogenesis and immunoprotection is the Gal/GalNAc inhibitable lectin, possessed by both pathogenic (Entamoeba histolytica) and nonpathogenic (Entamoeba dispar) strains, but only E. histolytica infection is associated with an acute inflammation and subsequently the disease. RESULTS AND DISCUSSION: The stimulation with the lectin induces the secretion of various proinflammatory cytokines/chemokines from intestinal epithelial cells. The differential induction of cytokine/chemokine network by the two strains can further regulate the immunoregulatory functions of the immune cells (monocytes and neutrophils) of the host. The soluble levels of IL-1beta, IL-6, IL-8, IL-10, MIP-1alpha, MCP-1, RANTES, GRO-alpha, GMCSF were quantitated to be significantly higher in pathogenic lectin-induced conditioned media (LCM) compared to nonpathogenic LCM (NP-LCM). The monocytes from ALA patients responded to the presence of pathogenic-LCM (P-LCM) by lowering of intracellular Ca(2+) (which was still higher than control). The proinflammatory MCP-1, GRO-alpha, and GMCSF levels in the monocytes were recorded both (quantitatively and mRNA quantitation) to be tremendously higher along with their respective receptors, with P-LCM compared to NP-LCM. A significant increase in the reactive oxygen intermediates and chemotactic index (CI) was observed in the monocytes when treated with P-LCM. Similarly, in neutrophils of ALA patients, an increase in intracellular Ca(2+), ROS and chemotaxis was observed with P-LCM. OBJECTIVES: The study is a step towards understanding the mechanism of immunopathogenesis of amoebiasis, on one hand, and points to the central role of cytokine/chemokine network in the process, on the other hand.

Erythropoietin and its derivative protect the intestine from severe ischemia/reperfusion injury in the rat.

OBJECTIVE: To investigate the protective effect of erythropoietin (EPO) and its nonhematopoietic derivative (asialoEPO) against intestinal ischemia/reperfusion (I/R) injury in a rat model. METHODS: The superior mesenteric artery of Wistar rats was clamped for 60 minutes and then released. The rats were divided into 4 groups (n = 15 in each group): sham operation (Sham), vehicle treatment (Vehicle), EPO treatment (EPO), and asialoEPO treatment (AsialoEPO). EPO and asialoEPO were administered subcutaneously at 1000 units/kg for 10 minutes before clamping, 30 minutes after the start of clamping, and just before declamping. This treatment was followed by determination of 72-hour survival rates, serum TNF-alpha and IL-6 levels, histologic evaluation of the small intestine, quantification of the number of apoptotic cells, and analysis of the antiapoptotic molecules Bcl-xL and XIAP by Western blotting. RESULTS: The survival rates at 72 hours after I/R injury in the Sham, Vehicle, EPO, and AsialoEPO groups were 100%, 33%, 75%, and 83%, respectively (P < .05). Blood TNF-alpha and IL-6 were significantly more suppressed in the EPO and AsialoEPO groups than in the Vehicle group at 6 hours after I/R injury. Histologically, injury to villi in the EPO and AsialoEPO groups was significantly less than in the Vehicle group. The number of apoptotic cells in the EPO and AsialoEPO groups was significantly less than in the Vehicle group. Western blotting revealed that EPO and asialoEPO constitutively increased the expression of Bcl-xL. CONCLUSIONS: EPO and asialoEPO exert a strong protective effect against intestinal I/R injury, possibly by inhibiting release of TNF-alpha and IL-6 and decreasing apoptosis.

Asialoerythropoietin prevents contrast-induced nephropathy.

Strategies to prevent contrast-induced nephropathy (CIN) are suboptimal. Erythropoietin was recently found to be cytoprotective in a variety of nonhematopoietic cells, so it was hypothesized that the nonhematopoietic erythropoietin derivative asialoerythropoietin would prevent CIN. Nephropathy was induced in rats by injection of the radiocontrast medium Ioversol in addition to inhibition of prostaglandin and nitric oxide synthesis. Administration of a single dose of asialoerythropoietin before the induction of nephropathy significantly attenuated the resulting renal dysfunction and histologic renal tubular injury. Contrast-induced apoptosis of renal tubular cells was inhibited by asialoerythropoietin both in vivo and in vitro, and this effect was blocked by a Janus kinase 2 (JAK2) inhibitor in vitro. Furthermore, phospho-JAK2/signal transducer and activator of transcription 5 (STAT5) and heat-shock protein 70 increased after injection of asialoerythropoietin, suggesting that the effects of asialoerythropoietin may be mediated by the activation of the JAK2/STAT5 pathway. Overall, these findings suggest that asialoerythropoietin may have potential as a new therapeutic approach to prevent CIN given its ability to preserve renal function and directly protect renal tissue.