Susceptibility of corneal allografts and xenografts to antibody-mediated rejection

The effects of passive transfer of antisera containing cytotoxic antibodies to allo- and xenoantigens on survival of corneal allografts and xenografts were evaluated in experimental models. Corneas from allogeneic B10 or xenogeneic rat Lewis donors were grafted orthotopically into BALB/c mice. Recipient mice were treated with donor-specific antisera administered at the period of grafting or at 2 weeks after transplantation. Rejection was determined by the severity of corneal opacity using a standard scoring system. Treatment of graft recipients with donor-specific antisera accelerated the onset of graft rejection and significantly shortened survival times of both corneal allografts and xenografts. Corneal xenografts, which had been accepted after treatment with anti-CD4 monoclonal antibody, were acutely rejected by the passive transfer of antiserum against xenoantigens. The results suggest that corneal grafts are vulnerable to antibody-dependent immunity and that cytotoxic antibodies against graft donor antigens can mediate rejection of both corneal allografts and xenografts.     

CD34+ corneal stromal cells are bone marrow-derived and express hemopoietic stem cell markers

Previous studies have suggested that corneal stromal keratocytes express the CD34 antigen. We wished to investigate CD34 antigen expression in normal mouse cornea using dual- and triple-staining techniques. Whole-mount preparations of mouse and rat corneas were examined with confocal microscopy using single, dual, or triple immunostaining to study their morphology, phenotype, and distribution. Single-cell suspensions from normal mouse corneas were also prepared and analyzed by flow cytometry. After short-term culture of corneal stromal explants, nonadherent cells were harvested and cytospins were prepared and stained for different markers.Combined staining for F-actin and leukocyte differentiation markers clearly showed that the corneal stroma contains a population of CD45(+) resident bone marrow-derived cells, whereas most cells were CD45-F-actin(+) keratocytes. A significant proportion (two thirds) of CD45(+) cells in the normal corneal stroma expressed CD34(+), whereas no CD45(-) cells (i.e., keratocytes) coexpressed CD34. In addition, CD34(+) cells were CD11c(-) and CD11b(+). Fewer than 10% of the CD34(+) cells also coexpressed Sca-1(+), but no CD34(+) cells coexpressed major histocompatibility complex (MHC) class II(+). In contrast, the remaining population of CD45(+)CD34(-) cells in the corneal stroma expressed CD11b, MHC class II(+) but not CD11c and were found mostly in the anterior and peripheral part of stroma. These cells are in intimate contact with corneal keratocytes, which stained only for F-actin and were negative for all leukocyte markers. Very few CD45(+) cells expressed the B220 marker, suggesting a plasmacytoid dendritic cell phenotype. Flow cytometry analyses confirmed the morphometric data showing that 68% of CD45(+) cells coexpress CD34 and CD11b, whereas 22% are CD11b(+)CD34(-).We conclude that the normal mouse cornea contains two populations of bone marrow-derived leukocytes, both of which are distinct from stromal keratocytes. The larger population resembles CD34(+) hemopoietic stem cells, whereas the smaller population are CD34(-)CD11b(+) MHC class II(+) macrophages. A very small percentage comprises plasmacytoid dendritic cells.     

Ultrastructural features of the spermatozoon and its differentiation in Brandesia turgida (Brandes, 1888) (Digenea,Microphalloidea, Pleurogenidae)

Spermatological characters of the digenean Brandesia turgida (Brandes, 1888), an intestinal parasite of the frog Pelophylax ridibundus (Pallas, 1771), have been investigated by means of transmission electron microscopy for the first time. The process of the spermatozoon formation begins with the appearance of the differentiation zone bordered by cortical microtubules and containing two centrioles associated with striated rootlets and with an intercentriolar body. The intercentriolar body is made up of seven distinct electron-dense plates, two less electron-dense, and four electron-lucent zones. The orthogonal development of the two flagella is followed by a flagellar rotation and their proximodistal fusion with the median cytoplasmic process. This process is accompanied by an extension of both the mitochondrion and nucleus into the median cytoplasmic process. The mature spermatozoon of B. turgida contains two parallel axonemes of unequal lengths with the 9?+?“1” trepaxonematan pattern, mitochondrion, nucleus, parallel cortical microtubules, four electron-dense attachment zones, an external ornamentation of the plasma membrane, and electron-dense glycogen granules. The anterior extremity of the male gamete contains one complete centriole, a small component of the central element of the second centriole, and peripheral cortical microtubules (up to 45). The posterior extremity of the mature spematozoon exhibits tubular elements of the disorganized axoneme. The present study provides the first data on spermiogenesis within the family Pleurogenidae. Variations of the spermatozoa ultrastructural characters within Digenea, in particular, between different families of the superfamily Microphalloidea, are discussed.     

Vitellogenesis in two spathebothriidean cestodes

Vitellogenesis in two spathebothriidean tapeworms, dixenous adult Cyathocephalus truncatus and monoxenous progenetic Diplocotyle olrikii, has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. Each vitelline follicle consists of vitellocytes at various stages of development and one irregularly shaped interstitial cell. Projections of the interstitial cell enclose the vitellocytes and extend as a cytoplasmic sheath on the follicular periphery. An outer thin fibrous layer (= extracellular lamina) covers the cytoplasmic sheath in C. truncatus, but lacks in D. olrikii. Maturing and mature vitellocytes contain vitelline material in the form of single small shell globules that gradually fuse and give rise to the large shell globule clusters. Morphology of shell globule clusters differs slightly in both species. In addition, single lamellar granules are present in the cytoplasm of vitellocytes of C. truncatus, but not in D. olrikii. Both electron lucent and electron dense lipid droplets are present in the vitellocytes of C. truncatus, whereas only electron dense lipids occur in D. olrikii. A single lipid droplet turns up occasionally in the nuclei of some of the vitellocytes of C. truncatus. The ultrastructural features of vitellogenesis in spathebothriideans resemble those reported previously in lower cestodes, especially in pseudophyllideans.     

Advanced glycation end products in hemodialyzed patients with diabetes mellitus correlate with leptin and leptin/body fat ratio

Advanced glycation end products (AGEs) and other carbonyl and oxidative stress compounds are supposed to play a critical role in the pathogenesis of several diseases and their complications, i.e., diabetes mellitus, diabetic retinopathy, atherosclerosis, and chronic renal failure. In the present investigation, we were interested in the relationship of AGEs in plasma to other prominent factors in the patients on chronic hemodialysis treatment-27 patients with diabetes mellitus, 35 patients without diabetes mellitus. AGE-group reactivity was estimated using a spectrofluorometric method (excitation 350 nm, emission 430 nm) and is expressed in arbitrary units (AU). We found significantly higher AGEs levels in diabetics than in non-diabetics on regular hemodialysis treatment both before (2.7 +/- 0.7 x 10(4) AU vs. 2.2 +/- 0.6 x 10(4) AU, p < 0.001) and after the dialysis session (2.3 +/- 0.5 x 10(4) AU vs. 1.8 +/- 0.7 x 10(4) AU, p < 0.005). AGEs were significantly reduced during hemodialysis in both groups of patients--by 15.4% in the diabetic go (p < 0.001) and by 17.3% in non-diabetics (p < 0.005). In the patients with diabetes mellitus, AGEs did not correlate with parameters of the glucose metabolism correction (blood glucose, HbA1c). We observed a significant correlation between AGEs and leptin (r = 0.48, p < 0.05) as well as the leptin/body fat ratio (r = 0.56, p < 0.05) only in hemodialyzed patients with diabetes mellitus. These findings suggest more detailed studies to identify the molecular links between carbonyl stress, i.e., advanced glycation end products, and leptin metabolism, sign of microinflammation and hypertension.     

The myocardial infarct size-limiting and antiarrhythmic effects of acyl-CoA:cholesterol acyltransferase inhibitor VULM 1457 protect the hearts of diabetic-hypercholesterolaemic rats against ischaemia/reperfusion injury both in vitro and in vivo

The study was designed to characterise the influence of a novel acyl-CoA:cholesterol acyltransferase inhibitor, VULM 1457, on the severity of myocardial ischaemia-reperfusion injury in a model of diabetes mellitus and hypercholesterolaemia induced by co-administration of streptozotocin and a high fat-cholesterol diet. We used Langendorff-perfused rat hearts to measure the size of myocardial infarction after 30 min of regional ischaemia, followed by a 2-h reperfusion period, and open-chest rats were exposed to 6 min of ischaemia and 10 min of reperfusion to analyse ventricular arrhythmias. In addition to the high fat-cholesterol diet, VULM 1457 was administered to the diabetic-hypercholesterolaemic rats for 5 days. Decreased plasma and liver cholesterol levels and a significantly reduced occurrence of ventricular fibrillation (29% vs. 100%, P<0.01), determined via the mean number and duration of episodes (0.6+/-0.4 and 2.1+/-1.4 s vs. 2.8+/-0.8 and 53.5+/-14.4 s in diabetic-hypercholesterolaemic rats, both P<0.01), were observed in these animals. Lethal ventricular fibrillation was suppressed, and arrhythmia severity was also significantly decreased in these animals as compared to the non-treated animals (2.9+/-0.6 vs. 4.9+/-0.2; P<0.05). A smaller infarct size, normalised to the size of area at risk, was observed in the treated diabetic-hypercholesterolaemic group as compared to the non-treated group (16.3+/-1.9% vs. 37.3+/-3.1%; P<0.01). Aside from remarkable hypolipidaemic activity, VULM 1457 improved the overall myocardial ischaemia-reperfusion injury outcomes in the diabetic-hypercholesterolaemic rats by suppressing arrhythmogenesis as well as by reducing myocardial necrosis.     

Skin permeation of acyl derivatives of stobadine

Stobadine is a model drug with pyridoindole structure with cardioprotective and antioxidant properties. Permeation properties of its acyl derivatives were studied to find a proper prodrug form. The experimental study of transdermal delivery of derivatives was combined with the theoretical approach in which the partition coefficients of substances were estimated by means of fragmentation or quantum chemical calculations. All modes applied showed differences in the transport properties of derivative S1 (N-acetyl stobadine). The experimental results obtained for the flux of S1 were higher by one order of magnitude than for the other derivatives and the parent drug. The results are discussed from the point of view of physicochemical properties of derivatives. We concluded that the exceptional permeation value of S1 derivative results from the concurrence of partitioning coefficient, dissociation constant, and arrangement of permeation set.     

Ultrastructure of the vitellarium in the digeneans Phyllodistomum angulatum (Plagiorchiida, Gorgoderidae) and Azygia lucii (Strigeida, Azygiidae)

Fine structural features of the vitellarium of two digeneans, Phyllodistomum angulatum and Azygia lucii, are documented and compared with those of other digenean species. The cytodifferentiation of immature vitelline cells (vitellocytes) assumes the production and subsequent accumulation in their cytoplasm of several inclusions. Mature vitelline cells of P. angulatum are characterized by the presence of vitelline clusters (～2.7 μm in diameter, with ～100 vitelline globules of ～0.35 μm in diameter) and osmiophobic, saturated lipid droplets (～2-3 μm in diameter), and in A. lucii vitelline clusters of the same diameter include much fewer vitelline globules (～50 globules of ～0.5 μm in diameter), osmiophilic lipid droplets and α-glycogen. In both P. angulatum and A. lucii, interstitial cells are also present within the vitellarium. Two types of contact sites (septate and tight junctions) between adjoining interstitial cells also occur in both digenean species. Judging from the present and previous ultrastructural studies, it is suggested that there are three potential discriminatory characters of the digenean vitellarium (the number of different types of cell components within the vitellarium, the presence and type of junctional complexes between these cells, and the isolation of the vitellarium from the surrounding tissue) which may prove useful for a better understanding of the biology and evolutionary history of the different digenean groups.     

Early intrauterine embryonic development in <Emphasis Type="Italic">Khawia sinensis</Emphasis> Hsü, 1935 (Cestoda,Caryophyllidea, Lytocestidae), an invasive tapeworm of carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>): an ultrastructural study

Intrauterine embryonic development in the caryophyllidean tapeworm Khawia sinensis has been investigated using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. Contrary to previous light microscopy findings that reported the release of non-embryonated eggs of K. sinenesis to the external environment, the present study documents various stages of embryonation (ovoviviparity) within the intrauterine eggs of this cestode. At the initial stage of embryonic development, each fertilised oocyte is accompanied by several vitellocytes that become enclosed within the operculate, electrondense shell. Cleavage divisions result in formation of blastomeres (up to about 24 cells) of various sizes. Mitotic divisions and apparent rosette arrangment of the blastomeres, the latter atypical within the Eucestoda, are observed for the first time in the intrauterine eggs of K. sinenesis. The early embryo enclosed within the electrondense shell is surrounded by a thin membraneous layer which in some enlarged regions shows presence of nuclei. Simultaneously to multiplication and differentiation, some of the blastomeres undergo deterioration. A progressive degeneration of the vitellocytes within eggs provides nutritive reserves, including lipids, for the developing embryo. The possible significance of this atypical timing of the intrauterine embryonic development to (1) the ecology of K. sinensis and that of a recent introduction of another invasive tapeworm, the caryophyllidean Atractolytocestus huronensis Anthony, 1958 to Europe; and (2) the affiliation of caryophyllideans with other lower cestodes, are discussed.     

Spermatozoon cytoarchitecture of Amphilina foliacea (Platyhelminthes, Amphilinidea)

The mature spermatozoon of Amphilina foliacea Rudolphi, 1819 has been examined using transmission electron microscopy. The male gamete is filiform and tapered at both extremities. Its moderately electron-dense cytoplasm possesses two parallel axonemes of unequal lengths with the 9?+?“1” trepaxonematan pattern, a mitochondrion, a nucleus, parallel cortical microtubules, four electron-dense attachment zones, and electron-dense glycogen granules. A crested body is absent. The anterior extremity of the cell exhibits a single axoneme. The anteriormost cortical microtubules have been observed with the appearance of the second axoneme. The number of cortical microtubules reaches a maximum (up to 25) in the nucleated region III of the spermatozoon. A single mitochondrion extends from the middle of region II to the end of region III of the cell. Both axonemes have become disorganized in a similar way: the axonemal doublets disappear first, followed by the central core. The nucleus is surrounded by a few cortical microtubules in the proximal part of region V. In the distal extremity of the mature spermatozoon, there is only the nucleus. Differences of spermatozoon ultrastructure within Amphilinidea and other Neodermata are discussed.