Efficacy of human recombinant erythropoietin plus IFN-alpha in patients affected by chronic hepatitis C.

Determination of serum iron levels in patients affected by chronic hepatitis C is considered fundamental for studying the response to interferon-alpha (IFN-alpha) treatment. IFN could induce anemia, which is promptly corrected by exogenous administration of recombinant human erythropoietin (rHuEPO). The aim of our study was to verify the possible beneficial effect of rHuEPO in patients affected by chronic hepatitis C and treated with IFN. Seventy consecutive patients (42 males and 28 females, mean age 46.4+/-5.2 years) affected by chronic hepatitis C were enrolled. In all patients, chronic hepatitis C was diagnosed on the basis of clinical and biological findings (alanine aminotransferase [ALT] serum levels at least 2-fold higher than normal values for at least 12 months and the presence of anti-HCV antibodies). All patients were negative for hepatitis B virus (HBV) infection, hepatitis D virus (HDV infection, and HIV infection. Statistical analysis was carried out using the Wilcoxon nonparametric sum rank test, the Spearman correlation rank test, and the Friedman ANOVA and Kendall coefficient of concordance. At the end of the treatment, our study series showed significant differences in serum levels of AST (p < 0.001), iron (p < 0.001), and ferritin (p < 0.001). At the end of the follow-up period, significant differences were seen in ALT, aspartate (AST), and iron ferritin and transferrin levels. All differences favored patients who received IFN-alpha and rHuEPO. We think that the depletion of circulating iron may improve the immune response impaired by iron accumulation in the liver. Our study confirms the important role played by iron in the response to IFN treatment, suggesting that the use of rHuEPO induces a better response to IFN in patients with chronic hepatitis C by activation of erythropoiesis.     

Vascularization and tissue infiltration of a biodegradable polyurethane matrix

Urethanes are frequently used in biomedical applications because of their excellent biocompatibility. However, their use has been limited to bioresistant polyurethanes. The aim of this study was to develop a nontoxic biodegradable polyurethane and to test its potential for tissue compatibility. A matrix was synthesized with pentane diisocyanate (PDI) as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix. The matrix was biodegradable in an aqueous solution at 37 degrees C in vitro as well as in vivo. The polymer was mechanically stable at body temperatures and exhibited a glass transition temperature (Tg) of 67 degrees C. The porosity of the polymer network was between 10 and 2000 microm, with the majority of pores between 100 and 300 microm in diameter. This porosity was found to be adequate to support the adherence and proliferation of bone-marrow stromal cells (BMSC) and chondrocytes in vitro. The degradation products of the polymer were nontoxic to cells in vitro. Subdermal implants of the PDI-sucrose matrix did not exhibit toxicity in vivo and did not induce an acute inflammatory response in the host. However, some foreign-body giant cells did accumulate around the polymer and in its pores, suggesting its degradation is facilitated by hydrolysis as well as by giant cells. More important, subdermal implants of the polymer allowed marked infiltration of vascular and connective tissue, suggesting the free flow of fluids and nutrients in the implants. Because of the flexibility of the mechanical strength that can be obtained in urethanes and because of the ease with which a porous microtexture can be achieved, this matrix may be useful in many tissue-engineering applications.     

A comparative study of [3H]thymidine and [125I]iodo-2'-deoxyuridine uptake as indicators of in vivo cell proliferation induced by graft-versus-host reaction in the mouse

Since [3H]thymidine ([3H]TdR) and [125I]iododeoxyuridine ([125I]UdR) appear to label very different cell populations in different tissues, we carried out the present investigation to determine the value and limitations of the use of [125I]UdR as a DNA precursor in a study of grafted cell proliferation during the course of a graft-versus-host reaction in mice. Results showed that the iodine radioisotope was as efficient as [3H]thymidine when endogenous thymidylate synthetase was inhibited by fluorodeoxyuridine. The use of 125I as an in vivo labelling compound constitutes a simple technique for evaluating DNA synthesis, provided the unfixed labelled iodine is eliminated by in vitro ethanol washing of tissues. The optimal conditions for elution are defined but may vary from one tissue to another. [125I]UdR and [3H]TdR give similar pictures of the very intricate phenomena accompanying a chronic graft-versus-host reaction (GVHR) induced in mice by minor histoincompatible antigens.     

The extracellular matrix of healthy and atretic mouse ovarian follicles studied by lanthanum nitrate and polycation ruthenium red

Changes occurring in the fine structure of Zona pellucida and follicular fluid in growing and atretic follicles in mice were examined by electron microscopy using lanthanum nitrate and ruthenium red. The results showed that follicular fluid has a dense globular aspect in growing follicles but becomes more dispersed and globular-fibrillar in atretic follicles. It is formed by rounded negatively charged particles (20 nm in diameter) that are closely packed in growing follicles, but spaced and united by filaments in atretic follicles. These changes may be related to a changed role of granulosa cells and also to an altered passage of nutritive substances from the plasma into the follicle. The Zona pellucida is intensely colorable only in the atretic follicles, where it is composed of a dense globular fibrous matrix, but not in growing follicles, where it reveals a weak reaction with ruthenium red. While in the Zona pellucida of growing follicles the globular negatively charged particles (20 nm in diameter) probably due to sialic acid moieties are rarely evident, these particles become highly numerous and connected by microfilamentous structures in atretic follicles. The altered electrical charge of polyanions may reflect a different chemical permeability of the Zona pellucida. Then the increase of negative charged particles in the zona may have a role in a process of sequestration of the oocyte during atreasia.     

Intracellular lectin-binding sites in symbiont-bearingCrithidia species

Crithidia oncopelti, C. deanei, andC. desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Direct and indirect lectin-gold labeling techniques were used at the electron microscopic level in Lowicryl K4M-embedded cells to demonstrate the presence of intracellular lectin-binding sites. We used the lectinsUlex europaeus I, Griffonia simplicifolia II, Ricinus communis I, Arachis hypogaea, G. simplicifolia I, Wistaria floribunda, Limulus polyphemus, andCanavalia ensiformis, which recognize -l-fucose, - and -N-acetylglucosamine, -galactose and -N-acetylgalactosamine, -galactose, -galactose, -N-acetylgalactosamine, sialic acid and -d-mannose, and -d-glucose residues, respectively. The nucleus was the cellular structure most frequently labeled by the lectins. The Golgi complex was seldom labeled, whereas the endoplasmic reticulum and the flagellar pocket presented a large number of binding sites. Symbionts had their two unit membranes weakly labeled by the different lectins but displayed no labeling of the space between the membranes.     

Intertubular capillary changes in kidney allografts: a morphologic investigation on 61 renal specimens.

A total of 61 kidney allograft specimens (51 biopsies and 10 nephrectomies) from 44 patients were investigated. Transplant glomerulopathy (TGP) was found in 38 specimens. Electron microscopy disclosed peculiar intertubular capillary changes (ITCC) in 47 specimens (all those showing TGP and nine without TGP). They were characterized by splitting and multilayering of the basement membranes. TGP and ITCC were graded and found mild (in 23 and 22 specimens), moderate (in 11 and 16 specimens), and severe (in four and nine specimens). Both worsened with time lapse from transplantation. When both were compared in single specimens, their grading was similar or, with a few exceptions, more severe for ITCC. TGP and ITCC were more frequent in biopsies with morphologic evidence of rejection and less frequent in patients treated with Cyclosporine A. Repeated biopsies and nephrectomies were studied in 10 patients and a somewhat more precocious appearance of ITCC than of TGP was found. Earlier and more severe ITCC were found in two patients who had had two transplants as compared to those with one. Morphologic similarities between TGP and ITCC suggest a common pathogenetic mechanism related to endothelial damage. The association to the histological evidence of rejection as well as the apparently protective activity of Cyclosporine A could support the involvement of an immune-mediated mechanism in agreement with what is reported in literature. The constant association of ITCC and TGP and its possible more precocious appearance enforce the diagnostic value of the former, which allows to infer the presence or the subsequent development of TGP even in biopsies where glomeruli are lacking.     

Changes of ovarian microvasculature in hCG stimulated rabbits. A scanning electron microscopic study of corrosion casts

The microvasculature of the ovarian cortex was studied by means of scanning electron microscopy of vascular corrosion casts in estrous and hCG stimulated rabbits in order to establish a better understanding of the structural as well as functional vascular changes which accompany the evolution of the luteofollicular complex. According to the various shape and size seven different morphological types of vascular plexuses corresponding respectively to antral follicles (Types 1-2), atretic follicles (Types 3-4), periovulatory follicles (Type 5), growing pseudopregnant corpora lutea (Type 6) and regressing pseudopregnant corpora lutea (Type 7) were identified. Growing to mature cycling type follicles (Types 1-2) showed a gradual enlargement and proliferation of the theca capillaries. These changes, associated with capillary hyper-permeabilization were observed in ovulatory and post-ovulatory follicles (Types 5), after hCG stimulation. The corpus luteum formation (Types 6) was accompanied by additional capillary dilatation, diffuse angiogenetic sprouts and organization of a more conspicuous venous drainage. The regression of the corpus luteum (Type 7) was characterized by the appearance of avascular areas within the glandular tissue and by regression of vascular dilatation. The atretic follicle (Types 3-4) wall showed large interruptions (avascular areas) and focal invasion of the central cavity by newly formed capillaries randomly arranged. The hCG stimulation did not affect consistently the atretic follicle microvasculature. The present observation shows that both thecal capillary vasodilatation and angiogenetic processes support the gradual increase of ovarian blood flow during follicle growth and corpora lutea formation and that microvascular changes of atretic follicles are possibly related to a type of inflammatory reaction since they seem to be a consequence rather than a primary cause of atresia.     

Variation of the spatial position computed by Roentgen Stereophotogrammetric Analysis (RSA) under non-standard conditions

Roentgen Stereophotogrammetric Analysis (RSA) has been applied to different kinds of research in order to obtain important information in the biomechanics field. Operative requirements change according to the type of investigation and sometimes practical conditions do not always permit one to respect standard specifications. The aim of this paper is to verify the reliability of the system under non-standard conditions, studying the effects of the focus-to-film distance on the determination of 3D co-ordinates of markers. The application of statistical analysis, consisting of two and three way ANalysis Of VAriance (ANOVA), showed that focus-to-film distance is not a significant source of variation, i.e., the system can correctly compute the marker position inside the calibrated space under all conditions of magnification. Since we obtained a fluctuation due to focal distance change of 10(-5) mm (LSD for 95% confidence level), we can assert that this source causes a variation strictly within the limits required by clinical investigations. Thus, this study contributes to improving the flexibility of RSA in clinical applications, making the technical requirements of the radiographic set-up less constraining.     

Recombinant congenic strains of mice from B10.D2 and DBA/2: Their contribution to behavior genetic research and application to audiogenic seizures

Recombinant congenic strains (RCS) represent a series of related strains, each of which carries a small fraction of the genome of one strain (donor strain) on the genetic background of another strain (background strain). Recombinant inbred strains (RIS) are commonly used to identify major gene segregation and linkage and associations between behavior and quantitative trait loci, whereas recombinant congenic strains (RCS) open other complementary leads. The variability in the reactivity of RCS to a trait is thus the expression of few minor-effect genes originating from the donor strain, because the probability that major genes are present in any one RCS is low. Unlike RIS in which minor-effect genes are often masked by major genes, RCS enable the effects of minor genes to be studied. With our method, for a given trait, an estimate can be made of the gene strength distribution as well as an estimate of the minimal number of genes involved having a certain strength.This study was supported by the Centre National de la Recherche Scientifique (URA 1924 and CSEAL-UPS 44, CNRS), Université René-Descartes, Paris V UFR Biomédicale, and the Fondation pour la Recherche Médicale.     

Succinyl derivatives of N-tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid: their effects on the frog neuromuscular junction.

Succinic anhydride (SA) dissolved in Ringer solution buffered with N-tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid (SA-TES solution) potentiates the depolarizing action of acetylcholine (ACh, 10-40 microM) on frog muscle and the tension induced by bath application of this agonist. Applied from one side of a double-barrelled micropipette, SA-TES increases the amplitude of iontophoretically elicited ACh potentials. The potentiation of the effects of ACh by SA-TES does not involve changes in either the activity of the ACh esterase or the input resistance of the muscle membrane. For depolarizations of frog sartorius muscle, dose-response relationships obtained for ACh concentrations from 0.5 to 20 microM indicate that SA-TES increases the apparent affinity of ACh by a factor of 3. SA-TES exerts an "accelerating' effect on the responses elicited by bath-applied ACh; i.e., it increases the rate of depolarization when ACh is added to the bath and the rate of repolarization upon washing out. These effects are particularly marked in preparations treated with neostigmine (3 microM). SA-TES does not potentiate the depolarizing action of agonists which do not contain an ester group. Moreover, the time course of the responses elicited by these compounds is not influenced by SA-TES. SA-TES fails to influence significantly the effects of the neurally released transmitter. Only a 10% increase in the average amplitude of the endplate potentials was observed. SA hydrolyzes in about 30 min at room temperature; however the SA-TES solution retains its activity for several weeks. Succinate is inactive, and so is SA in Ringer buffered with phosphate. The SA-TES solution contains seven succinyl-TES derivatives, which were separated by ion-exchange chromatography and paper chromatography. At concentrations between 1 to 150 microM, these succinyl-TES derivatives affected the ACh-induced contraction of frog rectus abdominus muscle. The most abundant derivative potentiated the action of high doses of ACh, but was inhibitory at lower ones. The other derivatives were mostly inhibitory. These results are discussed in terms of two hypotheses. One postulates the presence of a diffusion barrier formed by groups that bind ACh and are saturated by SA-TES. The other assumes that SA-TES acts directly on the ACh receptor exerting its potentiating effect through a cooperative mechanism.