Regulatory CD4+ CD25+ T cells prevent thymic dysfunction in experimental chronic colitis.

Chronic colitis in T-cell deficient Tg epsilon26 mice develops due to a dysfunction of the thymus which generates colitogenic T cells after bone marrow (BM) transplantation. Regulatory CD4+ CD25+ T cells have been shown to prevent colitis in this model by normalizing the peripheral T-cell pool. We tested the hypothesis that T-cell normalization takes place in the thymus. Tg epsilon26 mice were transplanted with BM (BM-->Tg epsilon26 mice) and consequently received either CD4+ CD25+ or CD4+ CD25- cells from syngenic wild type mice. Furthermore, untransplanted Tg epsilon26 mice received CD4+ CD25+ or CD4+ CD25- cells or complete mesenteric lymph node cells. Transfer of regulatory. CD4+ CD25+ cells normalized the total number of thymocytes and the percentage and number of double positive CD4+ CD8+ cells in transplanted mice while percentage of single positive CD4+ and CD8+ thymocytes in BM-->Tg epsilon26 mice was reduced upon CD4+ CD25+ transfer. Timing of CD4+ CD25+ cell injection was important as transfer later than 7 days after BM transplantation failed to prevent abnormal thymic T-cell distribution in BM-->Tg epsilon26 mice. Isolated CD4+ CD25+ cell transfer without preceding BM transplantation failed to reconstitute thymic architecture. Differences of thymic cell composition could not be exclusively explained by presence or absence of colitis, respectively, because 19 days after BM transplantation when both groups showed no histological signs of colitis, animals transferred with CD4+ CD25+ T cells had a significantly higher percentage and number of CD4+ CD25+ thymocytes and CD4+ Foxp3+ cells than BM-->Tg epsilon26 mice. In conclusion, early CD4+ CD25+ cotransfer prevents thymic dysfunction which underlies immune-mediated bowel inflammation in BM-->Tg epsilon26 mice.

Plasma etching and ashing: a technique for demonstrating internal structures of helminths using scanning electron microscopy.

Plasma etching and ashing for demonstrating the three-dimensional ultrastructure of the internal organs of helminths is described. Adult worms of the cestode Caryophyllaeides fennica were dehydrated through an ethanol series, critical point dried (Polaron E3000) and sputter coated with 60% gold-palladium (Polaron E5100) and glued to a standard scanning electron microscope (SEM) stub positioned as required for ashing. After initial SEM viewing of worm surfaces for orientation, stubs were placed individually in the reactor chamber of a PT7150 plasma etching and ashing machine. Worms were exposed to a radio frequency (RF) potential in a low pressure (0.2 mbar) oxygen atmosphere at room temperature. The oxidation process was controlled by varying the times of exposure to the RF potential between 2 to 30 min, depending on the depth of surface tissue to be removed to expose target organs or tissues. After each exposure the oxidized layer was blown from the surface with compressed air, the specimen sputter-coated, and viewed by SEM. The procedure was repeated as necessary, to progressively expose successive layers. Fine details of organs, cells within, and cell contents were revealed. Ashing has the advantage of providing three dimensional images of the arrangement of organs that are impossible to visualize by any other procedure, for example facilitating testes counts in cestodes. Both freshly-fixed and long-term stored helminths can be ashed. Ashing times to obtain the desired results were determined by trial so that some duplicate material was needed.

Regulatory CD4+CD25+ cells reverse imbalances in the T cell pool of bone marrow transplanted TGepsilon26 mice leading to the prevention of colitis.

BACKGROUND AND AIMS: Erroneous thymic selection of developing T lymphocytes may be responsible for the expansion of self reactive T cells or may contribute to the absence of regulatory T cells important in controlling peripheral inflammatory processes. Colitis in bone marrow (BM) transplanted Tgepsilon26 mice is induced by abnormally activated T cells developing in an aberrant thymic microenvironment. We investigated the protective role of regulatory CD4+CD25+ T cells in this model. METHODS: BM from (C57BL/6 x CBA/J) F1 mice was transplanted into specific pathogen free Tgepsilon26 mice (BM-->Tgepsilon26). Transplanted mice received no cells (control), sorted CD4+CD25+, or CD4+CD25- cells from mesenteric lymph nodes (MLN) of normal mice. MLN cell subsets were analysed using membrane markers. Cytokine secretion of MLN cells was measured using intracellular cytokine staining and cytokine secretion in anti-CD3 stimulated cell cultures. Colitis was measured by histological scores. RESULTS: CD4+CD25+ cells were reduced in the MLNs of BM-->Tgepsilon26 mice. Transfer of regulatory CD4CD4+CD25+ but not of CD4+CD25- cells reduced the number of MLN CD4+ T cells in BM-->Tgepsilon26 recipients and increased the number of MLN CD8+ cells, thereby normalising the CD4+/CD8+ ratio. CD4+CD25+ but not CD4+CD25- cell transfer into BM-->Tgepsilon26 mice reduced the number of tumour necrosis factor alpha+ CD4+ cells and increased the secretion of transforming growth factor beta by MLN cells. Transfer of 3 x 10(5) CD4+CD25+ cells after BM transplantation into Tgepsilon26 mice prevented colitis whereas CD4+CD25- cells had no protective effect. CONCLUSIONS: These results suggest that defective selection or induction of regulatory T cells in the abnormal thymus is responsible for the development of colitis in BM-->Tgepsilon26 mice. Transfer of CD4+CD25+ cells can control intestinal inflammation in BM-->Tgepsilon26 mice by normalising the number and function of the MLN T cell pool.

[Symptomatic dissection of the internal carotid artery. A rare manifestation of autosome dominant polycystic kidney disease?]. / Symptomatische Dissektion der Arteria carotis interna. Eine seltene Manifestation der autosomal-dominanten polyzystischen Nierenerkrankung?

Autosomal dominant polycystic kidney disease (ADPKD) is a frequent, genetically heterogenous disease with renal and extrarenal manifestations. Intracranial aneurysms are found in about 10% of cases. Other vascular manifestations of ADPKD have been described only in small case series. We report a 44-year-old ADPKD patient who developed acute large middle cerebral artery infarction secondary to subpetrous dissection of the internal carotid artery. Six months after the stroke, pseudoaneurysm was demonstrated on magnetic resonance angiography at the site of a previous dissection. Based on this case report, we review the spectrum of neurovascular manifestations and stroke associated with ADPKD and summarize current concepts of the pathogenesis of this disease. Finally, special aspects of the diagnostic evaluation and therapeutic management in patients with ADPKD and cervicocephalic dissection are discussed.

Larval trypanorhynchs (Platyhelminthes: Eucestoda: Trypanorhyncha) from black-scabbard fish, Aphanopus carbo and oceanic horse mackerel, Trachurus picturatus in Madeira (Portugal).

Four different types of trypanorhynch postlarvae were found attached to the stomach mucosa, external stomach wall or free in the body cavity of two marine fishes from Madeira, namely blackscabbard fish, Aphanopus carbo and oceanic horse mackerel, Trachurus picturatus. Morphological features shown by SEM indicated that the postlarvae belonged to the species Tentacularia coryphaenae, Sphyriocephalus tergestinus, Nybelinia lingualis and possibly N. yamaguitii. Prevalence [mean intensity (range)] of T. coryphaenae, S. tergestinus and Nybelinia spp. in A. carbo (n = 135) was 12.6% [1.65 +/- 1.27(1-6)], 5.9% [1.57 +/- 0.79 (1-3)] and 2.2% [1.33 +/- 0.58 (1-2)] respectively. The prevalence of T. coryphaenae and S. tergestinus showed some seasonality, with a rise in prevalence of T. coryphaenae corresponding to a decrease in prevalence of S. tergestinus. However these differences were not significant. In T. picturatus (n = 304) only N. lingualis was found at a prevalence of 9.6%. Both S. tergestinus and N. lingualis were recovered only from the stomach mucosa or external stomach wall, while T. coryphaenae was observed either attached to the stomach mucosa or free in the visceral cavity of the fish. The paper presents the first scanning electron micrographs (SEM) of Sphyriocephalus tergestinus and a new geographical record of N. lingualis in T. picturatus.

Continuous stimulation by normal luminal bacteria is essential for the development and perpetuation of colitis in Tg(epsilon26) mice.

BACKGROUND & AIMS: Normal resident bacteria are required for development of colitis in several rodent models. We determined whether bacterial stimulation is necessary for both induction and perpetuation of mucosal inflammation and T-cell activation in Tg(epsilon26) mice, in which transplantation of wild-type bone marrow (BM-->Tg(epsilon26)) causes colitis under specific pathogen-free (SPF) conditions. METHODS: BM from (C57BL/6 X CBA/J) F1 mice was transplanted into germfree (GF) or SPF Tg(epsilon26) mice. Mesenteric lymph node (MLN) cells from these mice were then transferred into SPF or GF recipients. Colitis and activation of MLN cells were measured by histologic scores, membrane marker analysis, and intracellular cytokine staining. Cytokine secretion by MLN cells stimulated by anti-CD3 or by luminal or epithelial antigens was measured by ELISA. RESULTS: Colitis did not develop when BM was transferred into GF recipient mice (BM-->GF Tg(epsilon26)). T lymphocytes that secreted interferon gamma upon activation were present in the MLN of BM-->GF Tg(epsilon26) mice, albeit in lower frequency than in control BM-->SPF Tg(epsilon26) mice. Furthermore, transfer of MLN cells from BM-->SPF Tg(epsilon26) mice into SPF Tg(epsilon26) recipients induced active colitis, but not if the same cells were transferred into GF Tg(epsilon26) recipients. Although CD4 T cells were detected in the colonic mucosa of GF recipients, no inflammation was observed for at least 31 weeks. In a reciprocal experiment, MLN cells from BM-->GF Tg(epsilon26) mice without colitis transferred disease to SPF Tg(epsilon26) recipients within 2-4 weeks. CONCLUSIONS: Activated T cells are present in the mucosa of BM-->GF Tg(epsilon26) mice but are incapable of inducing disease unless colonic bacteria are present. Moreover, pathogenic T cells require the continuous presence of colonic bacteria to sustain colitis.

Cystacanths of Bolbosoma vasculosum in the black scabbard fish Aphanopus carbo, oceanic horse mackerel Trachurus picturatus and common dolphin Delphinus delphis from Madeira, Portugal.

Cystacanths of the acanthocephalan, Bolbosoma vasculosum Rudolphi 1819, were found to be encapsulated in the connective tissues of the viscera of the black scabbard fish, Aphanopus carbo and oceanic horse mackerel, Trachurus picturatusfrom Madeira, Atlantic Ocean. Juvenile worms were obtained from the intestine of a stranded common dolphin, Delphinus delphis, also from Madeira. Cystacanths were 11-15 mm long, with a proboscis of 18-19 longitudinal rows, eight hooks per row, and two sets of trunk spines. Overall, the morphology and dimensions of the proboscis, neck and trunk corresponded to previous descriptions. Scanning electron microscopy of the proboscis structures and trunk spines is provided for the first time. The prevalence of B. vasculosum in A. carbo increased with fish length. There were no statistical differences in the prevalence and intensity of infection between seasons. The intensity of infection was similar for male and female fishes, but there were significant differences in relation to length, longer fishes having heavier infections. Aphanopus carbo from Madeira represents a new host record and a new geographic location for B. vasculosum.

IL-2-deficient mice raised under germfree conditions develop delayed mild focal intestinal inflammation.

Interleukin-2 (IL-2) amplifies immune stimuli and influences B cell differentiation. IL-2-deficient mice spontaneously develop intestinal inflammation if raised under specific pathogen-free (SPF) conditions. We quantitatively determined the aggressiveness and kinetics of gastrointestinal and hepatic inflammation in the presence or absence of viable bacteria in IL-2-deficient mice. Breeding colonies were maintained under SPF and germfree (GF) conditions. Intestinal tissues, serum, and mesenteric lymph nodes were obtained from mice at different ages for blind histological scoring, immunoglobulin measurements, mucosal T cell infiltration, and cytokine secretion. GF IL-2 -/- mice developed mild, focal, and nonlethal intestinal inflammation with delayed onset, whereas the more aggressive inflammation in SPF IL-2 -/- mice led to their death between 28 and 32 wk. Periportal hepatic inflammation was equal in the presence or absence of bacterial colonization. Intestinal immunoglobulin secretion decreased significantly by 13 wk of age in IL-2 -/- mice in both GF and SPF environments. In contrast to other genetically engineered rodents, IL-2 -/- mice develop mild focal gastrointestinal and active portal tract inflammation in the absence of viable bacteria.

Freeze fixation-dehydration as a method of preparation of Gyrodactylus (Monogenea) for SEM.

Freeze fixation-dehydration was used for the first time in the preparation of attached Gyrodactylus for SEM viewing. The technique provided instant immobilization of specimens before death and negligible shrinkage throughout the fixation-dehydration process. Comparisons of sample means of two linear measurements of attached opisthaptors showed 20% more shrinkage of Gyrodactylus fixed using 10% neutral buffered formalin than those which were freeze fixed. Freeze fixation-dehydration was excellent for the study of gross external morphology of attached Gyrodactylus. However, the freeze fixation-dehydration process may cause disruption of intracellular structural components making delicate tissues brittle and more prone to damage during subsequent manipulation.

Soft X-ray contact imaging of nucleolar chromatin using synchrotron radiation: a comparative scanning and transmission electron microscope study.

Contact images (CI) of dehydrated, nucleolar chromatin from amphibian oocytes have been produced by soft X-rays from a synchrotron radiation source. These CI have been compared with the morphology of the original chromatin as seen in scanning and transmission electron microscopes. The quality and informational content of the CI depend very much on certain preparative procedures. The following factors have a marked effect on image quality and need to be carefully controlled: the total X-ray dose, the time and nature of development and the distance of the specimen from the photoresist. The preparation of the chromatin itself, providing that it is critically point dried, is less important. By following a regime of high X-ray dose, sufficient for penetration of the rather thick chromatin rings, and gentle development so that fine detail is not dissolved from the resist surface, it has been possible to obtain images which closely resemble the original chromatin, although the detailed resolution of the CI is not as clear. The smallest biological structures clearly resolved in the CI are ribonucleoprotein granules, which vary in size from 200 to 800 nm. However, by further refinement of preparative conditions it should be possible to improve on the informational content of these images.

Effects of calcium antagonists and calcium-buffered salines on wound healing in Xenopus early embryos.

The role of calcium in the process of wound closure in Xenopus early embryos was studied. Embryos were wounded in the presence of the calcium antagonists D-600 and TMB-8 or in calcium-buffered salines, and the effects on wound healing were observed by scanning electron microscopy. D-600 and TMB-8 inhibit wound closure and these antagonists appear to act synergistically since their combined effect is greater than their individual effects. Experiments with calcium-buffered salines suggest that wound closure can proceed in the presence of low extracellular calcium. In all conditions there is a correlation between the degree of wound closure and the shapes of the cells at the wound margin; closing wounds are accompanied by cells elongated radial to the wound, gaping (non-closing) wounds are accompanied by cells stretched tangential to the wound. Thus the results suggest that calcium influx may not be a requirement for the changes in cell shape which accompany, and probably effect, wound closure in Xenopus early embryos.

Growth and ultrastructure of Streptomyces venezuelae during chloramphenicol production.

Streptomyces venezuelae (3022a) was grown in flask cultures and fermentors, using three media having differential effects on chloramphenicol production. Micromorphology, ultrastructure and chloramphenicol concentrations were studied during the growth cycle in each medium. Chloramphenicol production was greatest in the glycerol-serine-lactate (GSL) medium, less in the glycerol-nutrient broth-yeast extract (GNY) medium and very low in glucose-mineral salts (GA) medium. In GSL and GA, much growth was in the form of microcolonies, especially in flask cultures, while short hyphal fragments predominated in GNY. The major ultrastructural features were the high frequency of mesosomes in fragmenting hyphae in GNY, and electron-transparent zones which appeared during chloramphenicol synthesis in GSL. None of the structural abnormalities induced by chloramphenicol in sensitive organisms were observed in S. venezuelae despite high levels of the antibiotic in GSL medium.