Podocyte changes after induction of acute albuminuria in mice by anti-aminopeptidase A mAb

Administration of a specific combination of anti-aminopeptidase A (APA) mAb (ASD-37/41) in mice induces an acute albuminuria which is independent of angiotensin II, a well-known substrate of APA. In the present experiments, we examined whether binding of the mAb initiated changes in the podocytic expression of cytoskeleton (-associated), adhesion and slit-diaphragm proteins in relation to the time course of albuminuria. In addition, we measured ultrastructurally the extent of foot process retraction (the number of foot processes per microm GBM) and the width of the slit pore between the podocytes by morphometric methods. An injection of the mAb combination ASD-37/41 induced a massive but transient albuminuria that started at 6 h, and peaked at 8 h, after which it declined. However, even at day 7 after injection of the mAbs some albuminuria was present. Injection of the combination ASD-3/41 or saline did not induce an albuminuria. Notably, we observed changes in the staining of CD2AP and podocin, two slit-pore-associated proteins that coincided with the start of the albuminuria. Nephrin staining was reduced and podocytic actin staining became more granular only at a time albuminuria was declining (24 h). The number of foot processes per microm GBM was already decreased at 4 h with a further reduction thereafter. The width of the slit pore was unchanged at the time of peak albuminuria and gradually decreased thereafter. At day 7, podocytic foot process effacement was even more prominent although albuminuria was only slightly abnormal. Expression of CD2AP was still granular. We observed however a change toward normal in the expression of podocin. Injection of saline or ASD-3/41 had no effect on the expression of podocytic proteins, the number of foot processes or width of the slit pore. Our data show that the onset of albuminuria in the anti-APA model is related to alterations in CD2AP and podocin, proteins that are important for maintaining slit-diaphragm structure and podocytic function. Extended studies at day 7 demonstrated uncoupling of albuminuria, podocytic foot process effacement and CD2AP staining. Changes in podocin more closely paralleled changes in albuminuria.     

Monoclonal antibodies to human cartilage oligomeric matrix protein: epitope mapping and characterization of sandwich ELISA

BACKGROUND: Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP 5) is one of the most promising serologic markers with regard to an ability to prognose development of osteoarthritis (OA). Our aim was to map the epitopes of three monoclonal antibodies (mAb) to COMP and to develop and characterize a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring COMP levels in human body fluids. METHODS: COMP was digested with trypsin and the NH(2)-terminal sequence of the fragments recognized by each of the mAbs was determined. Steric competition among the mAbs was tested with an antibody capture assay. A sandwich ELISA was developed using unlabeled mAb 16-F12 as a capture antibody, and mAb 17-C10 labeled with biotin as the second antibody. RESULTS: Epitopes of the three mAbs were mapped to three different domains within the COMP subunit (16-F12, NH(2)-terminal domain; 17-C10, EGF-like domain; 12-C4, COOH-terminal domain). These epitopes did not overlap. mAbs 17-C10 and 12-C4 yielded similar serum COMP results when used as the secondary antibodies. Serum COMP levels measured with the new sandwich ELISA using mAbs 16-F12 and 17-C10 correlated strongly with results based on an inhibition ELISA with mAb 17-C10 alone (r(2) = 0.836; P < 0.0001). We characterized the new sandwich ELISA with regards to inter- and intra-assay variability, the range of COMP levels that can be expected in human synovial fluids (SF) and sera (controls and OA and rheumatoid arthritis (RA) patients), and the day-to-day and diurnal variability of COMP levels in sera. CONCLUSIONS: We have developed and characterized a sandwich ELISA for COMP that is sensitive and yields highly reproducible COMP results upon analysis of human sera and synovial fluids.     

Biliary pronucleating proteins and apolipoprotein E in cholesterol and pigment stone patients

BACKGROUND/AIMS: Although cholesterol gallstone patients exhibit higher biliary cholesterol saturation than pigment stone patients, underlying mechanisms that affect stone type are unknown. We hypothesized that pronucleating proteins, hydrophobic bile salts or apolipoprotein E genotype affect stone type. We therefore compared these putative factors in cholesterol and pigment stone patients.METHODS: In 74 cholesterol and 12 pigment stone patients, bile lipids, various pronucleating proteins, crystallization and apolipoprotein E genotype were determined.RESULTS: Crystallization was enhanced, and cholesterol saturation higher in case of cholesterol stones, without any difference in bile salt composition. Concentrations of mucin (0.91+/-0.08 versus 0.31+/-0.06 mg/ml: P<0.0001), protein, IgM, IgG, IgA, haptoglobin, alpha1-acid glycoprotein and haptoglobin were 2-6-fold higher in cholesterol stone patients. Twenty cholesterol stone pts (27%) but only one pigment stone pt (8%) had at least one epsilon4 allele. There was a significant difference in allele frequencies between both groups (cholesterol stones similar to Dutch population: epsilon2 0.074, epsilon3 0.770, epsilon4 0.156: pigment stones: epsilon2 0.250, epsilon3 0.708, epsilon4 0.042).CONCLUSIONS: Various pronucleating biliary proteins are markedly higher in cholesterol than pigment stone patients. Also, apolipoprotein E genotype differs between cholesterol and pigment stone patients. These factors may affect gallstone type.     

Reconstruction of epidermis by grafting of keratinocytes cultured on polymer support--clinical study

BACKGROUND: Extensive wound coverage still represents a challenge for contemporary medicine. We demonstrate the results of a clinical trial of the grafting of cultured keratinocytes directly on a polymer cultivation support in the treatment of skin defects in seriously burned patients and in patients with trophic ulcers. METHODS: Wound closure was evaluated clinically. The morphology and phenotypic pattern of the reconstructed epidermis, including the basal lamina, as well as the presence of Langerhans cells, were evaluated immunocytochemically using a panel of monoclonal antibodies. RESULTS: All layers of the reconstructed epidermis were normally differentiated (cytokeratin immunocytochemistry). The basal lamina contained collagen type IV and laminin. The reconstructed epidermis was extensively colonized by Langerhans cells. CONCLUSIONS: The results of the described technology are encouraging, especially in patients after a burn injury. The described procedure is suitable for the treatment of skin defects in clinical practice.     

Non-surfactant nanospheres of progesterone inclusion complexes with amphiphilic beta-cyclodextrins

Amphiphilic beta-cyclodextrins were formulated as nanospheres and characterised by particle size, zeta potential and TEM following freeze-fracture. The nanospheres were loaded with progesterone with different loading techniques involving the spontaneous formation of nanospheres from pre-formed inclusion complexes of amphiphilic beta-cyclodextrins modified on the primary or secondary face with progesterone. Inclusion complexes were characterised with various techniques including Differential Scanning Calorimetry (DSC), Fast Atom Bombardment Mass Spectrometry (FAB MS) and 1H NMR spectroscopy; and progesterone was believed to be partially included in the CD cavity. Loading properties of conventionally-loaded nanospheres were compared with those prepared directly from pre-formed inclusion complexes and loading technique was found to enhance associated drug percentage significantly (P<0.05). Although both amphiphilic beta-cyclodextrins (6-N-CAPRO-beta-CD and beta-CDC6) were capable of high progesterone loading, beta-CDC6 displayed slightly higher entrapment efficiency due to the possible higher affinity of progesterone to the 14 alkyl chains surrounding this molecule resulting in higher drug adsorption to particle surface. Progesterone was released within a period of 1 h from all formulations. Progesterone-loaded amphiphilic beta-CD nanospheres were proved to be a promising non-surfactant injectable delivery system providing high-quantity of water-insoluble progesterone rapidly within 1 h.     

Effects of N-acetyl-glucosamine-coated glycodendrimers as biological modulators in the B16F10 melanoma model in vivo

Glyco-coat changes on cancer cells due to aberrant glycosylation are potential targets for immune recognition through lectin-like receptors on immune cells. These cells include natural killer (NK), CD8+ and CD4+ lymphocytes, all reported to have, together with cytokines, important functions in antitumor immunity. The aim of this study was to evaluate a possible role of synthetic monodisperse multivalent neo-glycoconjugates, namely glycodendrimers, as a new approach to anticancer immune modulation through carbohydrate-mediated immune recognition. Octavalent polyamidoamine dendrimers functionalized with N-acetyl-glucosamine residues (PAMAM-GlcNAc8), with in vitro high affinity for the recombinant lymphocyte receptor NKR-P1A, were employed. To follow the fate of the compound, a fluorescent marker was conjugated to the tetra-branched semi-component of the dendrimer. Tumor development and immunity were evaluated in C57BL/6 mice. Animals were inoculated with B16F10 melanoma cells and underwent different protocols of PAMAM-GlcNAc8 administration. Advantages on survival and reduction of tumor growth were obtained in dose-dependent manner, by IP route. Increase of CD69+ cells in the spleen and their appearance inside the tumors, early progressive release of IL-1beta, a later production of INFgamma and IL-2 concomitant to an increment of CD4+ cells were observed. Cytotoxicity assays, performed ex vivo, showed an enhanced NK cell activity proportioned to the percentage of activated NK cells. Our data suggest that well-defined multivalent neo-glycoconjugates can stimulate an antitumor immune response engaging both innate and acquired immunity.     

Plasma lipoproteins in transport of silibinin,an antioxidant flavonolignan from Silybum marianum

To assess the role of plasma lipoproteins in the transport of silibinin, an antioxidant flavonolignan, (125)I-labelled silibinin ((125)I-SB) administered perorally to the rat was used. The plasma (125)I-SB derived radioactivity was distributed among plasma lipoproteins according to their lipophilicity (TAG-rich lipoproteins 30-40% > LDL 15% > HDL 5%), and in the fraction of d > 1.215 containing albumin and other proteins a minority amount of radioactivity was found. Administration of (125)I-SB in a complex with phosphatidylcholine resulted in proportionally higher radioactivities in all fractions as well as in tissues. Dietary olive oil had a slightly decreasing effect on plasma concentrations of silibinin measured by HPLC as well as on (125)I-SB derived radioactivity in plasma and liver. In the TAG-rich lipoprotein fraction and HDL no effects of olive oil on the levels of (125)I-SB derived radioactivities were observed, however, at a 30 min interval the levels of (125)I-SB derived radioactivity in LDL and the heart were significantly decreased in the olive oil group. These results suggest that (i) silibinin is not resorbed by the chylomicron pathway, and (ii) the endogenous lipoprotein pathway VLDL --> LDL may play a role in the transport of silibinin from the liver to the extrahepatic tissues concurrently facilitating the lipoprotein antioxidant influence of silibinin.     

An experimental evaluation of three preoperative radiation regimens for resectable rectal cancer

Background We investigated the degree of tumor cell killing after radiotherapy regimens commonly used in clinical practice in comparison with an accelerated schedule. Methods Mtln3 mammary adenocarcinoma tumor cells were inoculated subcutaneously in the hind leg of syngeneic Fischer 344 rats. Tumors were irradiated with 5×5 Gy in 5 days, 10×3 Gy over 10 days, or 5×(2×3) Gy in 5 days. After excision of the irradiated tumors, the dye exclusion, a tetrazolium-based colorimetric and the clonogenic assays were used to determine tumor cell viability and surviving fractions. Results Estimated potential doubling time values indicate a rapid proliferation capacity, comparable with potential doubling time values in human rectal cancer. The dye exclusion and clonogenic assays revealed a significantly higher degree of cell killing after the hypofractionated and the accelerated regimens of, respectively, 5×5 Gy and 5×(2×3) Gy over 5 days compared with 10×3 Gy over 10 days. Conclusions A shorter treatment time offered the best therapeutic efficacy. The schedule involving two daily fractions of 3 Gy over 5 days should be less toxic than 5×5 Gy and may therefore provide a therapeutic advantage.     

Role of magnesium in the reduction of ischemic depolarization and lesion volume after experimental subarachnoid hemorrhage

OBJECT: Ischemia-induced tissue depolarizations probably play an important role in the pathophysiology of cerebral ischemia caused by parent vessel occlusion. Their role in ischemia caused by subarachnoid hemorrhage (SAH) remains to be investigated. The authors determined whether ischemic depolarizations (IDs) or cortical spreading depressions (CSDs) occur after SAH, and how these relate to the extent of tissue injury measured on magnetic resonance (MR) images. In addition, they assessed whether administration of MgSO4 reduces depolarization time and lesion volume. METHODS: By means of the endovascular suture model, experimental SAH was induced in 52 rats, of which 37 were appropriate for analysis, including four animals that underwent sham operations. Before induction of SAH, serum Mg++ levels were measured and 90 mg/kg intravascular MgSO4 or saline was given. Extracellular direct current potentials were continuously recorded from six Ag/AgCl electrodes, before and up to 90 minutes following SAH, after which serum Mg++ levels were again measured. Next, animals were transferred to the MR imaging magnet for diffusion-weighted (DW) MR imaging. Depolarization times per electrode were averaged to determine a mean depolarization time per animal. No depolarizations occurred in sham-operated animals. Ischemic depolarizations occurred at all electrodes in all animals after SAH. Only two animals displayed a single spreading depression-like depolarization. The mean duration of the ID time was 41 +/- 25 minutes in the saline-treated controls and 31 +/- 30 minutes in the Mg++-treated animals (difference 10 minutes: p = 0.31). Apparent diffusion coefficient (ADC) maps of tissue H2O, obtained using DW images approximately 2.5 hours after SAH induction, demonstrated hypointensities in both hemispheres, but predominantly in the ipsilateral cortex. No ADC abnormalities were found in sham-operated animals. The mean lesion volume, as defined on the basis of a significant ADC reduction, was 0.32 +/- 0.42 ml in saline-treated controls and 0.11 +/- 0.06 ml in Mg++-treated animals (difference 0.21 ml; p = 0.045). Serum Mg++ levels were significantly elevated in the Mg++-treated group. CONCLUSIONS: On the basis of their data, the authors suggest that CSDs play a minor role, if any, in the acute pathophysiology of SAH. Administration of Mg++ reduces the cerebral lesion volume that is present during the acute period after SAH. The neuroprotective value of Mg++ after SAH may, in part, be explained by a reduction in the duration of the ID of brain cells.     

Benign cranial dural arteriovenous fistulas: outcome of conservative management based on the natural history of the lesion

OBJECT: Cranial dural arteriovenous fistulas (DAVFs) can be classified into benign or aggressive, based on their patterns of venous drainage. A benign condition requires the absence of cortical venous drainage (CVD). The clinical and angiographic features of a consecutive single-center group of 117 patients harboring benign cranial DAVFs were evaluated over time to validate the behavior and appropriate management of these lesions. METHODS: At the initial assessment four patients were asymptomatic. Two infants presented with congestive heart failure. All other patients presented with other benign symptoms: chronic headache, bruit, or orbital phenomena. Observational management was instituted in 73 patients (62%). Intolerable bruit or ophthalmological sequelae were deemed indications for palliative embolization in 43 patients and surgical treatment in one patient. A median follow-up period of 27.9 months (range 1 month-17.5 years) was available in 112 patients (95.7%), among whom repeated angiography was performed in 50. Overall, observational and palliative management resulted in a benign and tolerable level of disease in 110 (98.2%) of 112 cases. In two cases managed conservatively CVD developed. In both of these cases the conversion from benign to aggressive DAVF was associated with spontaneous progressive thrombosis of venous outlets. CONCLUSIONS: The disease course of a cranial DAVF without CVD is indeed benign, obviating the need for a cure of these lesions. Symptoms are well tolerated with either observation or palliative treatment. After a long-term follow-up review of 68 patients, this conservative management resulted in a benign and tolerable level of disease in 98.5% of cases. It is noteworthy, however, that a benign DAVF carries a 2% risk of developing CVD, mandating close clinical follow-up review in such cases and renewed radiological evaluation in response to any deterioration in the patient's condition.