Practice of dilatation after surgical correction in anorectal malformations.

BACKGROUND: In order to prevent stricture of the neoanus after surgical correction, regular dilatation is recommended. There is a lack of knowledge about the performance of anal dilatation and the occurrence of pain. The aim of our investigation was to describe the practice of dilatation and to identify possible risk factors for painful procedures. METHODS: Congenital Uro-Rectal Malformations Network is a German interdisciplinary multicenter research network. With standard questionnaires, physicians interviewed 243 patients and/or their parents at home, additional 103 patients born since 2009 were assessed through their treating physicians. RESULTS: In total, 88 % of the patients received dilatations. Treatment lasted for 7 months in median (range 1-156 months), until the age of 13 months (range 1-171 months). In 69 % painful dilatation was reported; without a significant differences in age or gender. In 32 % bleeding was reported. In 30 % at least one dilatation was performed under general anesthesia. In 11 % some kind of analgesia was used. Type of fistula, dilatations lasting longer than 10 months and Hegar size above 15 were relevant factors for experience of pain. There were about 16 % postoperative strictures of the neoanus, without reported differences in dilatation procedures; but there was a relation to type of malformation. CONCLUSION: Considering the high number of painful treatments, predictors for painful dilatations should be further clarified through standardized documentation and prospective evaluation in order to improve follow-up.

The circadian clock of fruit flies is blind after elimination of all known photoreceptors.

Circadian rhythms are entrained by light to follow the daily solar cycle. We show that Drosophila uses at least three light input pathways for this entrainment: (1) cryptochrome, acting in the pacemaker cells themselves, (2) the compound eyes, and (3) extraocular photoreception, possibly involving an internal structure known as the Hofbauer-Buchner eyelet, which is located underneath the compound eye and projects to the pacemaker center in the brain. Although influencing the circadian system in different ways, each input pathway appears capable of entraining circadian rhythms at the molecular and behavioral level. This entrainment is completely abolished in glass(60j) cry(b) double mutants, which lack all known external and internal eye structures in addition to being devoid of cryptochrome.

Ectopic expression of the neuropeptide pigment-dispersing factor alters behavioral rhythms in Drosophila melanogaster.

To study the function of the neuropeptide pigment-dispersing factor (PDF) in the circadian system of Drosophila, we misexpressed the pdf gene from the grasshopper Romalea in the CNS of Drosophila and investigated the effect of this on behavioral rhythmicity. pdf was either ectopically expressed in different numbers of neurons in the brain or the thoracical nervous system or overexpressed in the pacemaker neurons alone. We found severe alterations in the activity and eclosion rhythm of several but not all lines with ectopic pdf expression. Only ectopic pdf expression in neurons that projected into the dorsal central brain severely influenced activity rhythms. Therefore, we conclude that PDF acts as a neuromodulator in the dorsal central brain that is involved in the rhythmic control of behavior. Overexpression of pdf in the pacemaker neurons alone or in the other neurons that express the clock genes period (per) and timeless (tim) did not disturb the activity rhythm. Such flies still showed a rhythm in PDF accumulation in the central brain terminals. This rhythm was absent in the terminals of neurons that expressed PDF ectopically. Probably, PDF is rhythmically processed, transported, or secreted in neurons expressing per and tim, and additional PDF expression in these cells does not influence this rhythmic process. In neurons lacking per and tim, PDF appears to be continuously processed, leading to a constant PDF secretion at their nerve terminals. This may lead to conflicting signals in the rhythmic output pathway and result in a severely altered rhythmic behavior.

Loss of flk-2/flt3 expression during commitment of multipotent mouse hematopoietic progenitor cells to the mast cell lineage.

The expression of c-kit and flk-2/flt3 was analyzed in various stages of mast cell differentiation using reverse transcriptase polymerase chain reaction (RT-PCR). Mouse fetal liver cells were sorted using antibodies for Sca-1 (Ly6A/E) and CD43 to obtain a population enriched for early progenitors; committed mast cell progenitors were absent from this population. Mouse fetal liver-derived, IL-3-dependent blast cell colonies provided a source of committed mast cell progenitors, and mast cell colonies provided mature mast cells. Comparison of these populations showed that some uncommitted cells express both c-kit and flk-2/flt3. At the time of commitment to the mast cell lineage, the expression of c-kit increases compared to that of uncommitted progenitors, and the expression of flk-2/flt3 becomes undetectable. Previous studies have shown that steel factor, the ligand for c-kit, supports mast cell differentiation in vivo and in vitro. In contrast, the ligand for flk-2/flt3 is inactive on mast cells. Thus, receptor gene expression appears to be an important determinant of the response or lack of response of mast cells to the ligands for these two homologous receptors.

Depth segregation of retinal ganglion cells projecting to mouse superior colliculus.

The retinotectal projections in the mouse were analyzed with injections of horseradish peroxidase into the superior colliculus and of radioactive amino acids into the eye. At least 70% of the ganglion cells, and possibly all of them, were found to project to the superior colliculus, including ganglion cells of all sizes. Small injections revealed that ganglion cells of different sizes terminate at different levels in the superior colliculus. The small ganglion cells that form the vast majority of all cells project predominantly to the upper stratum griseum superficiale. A small population of mainly medium-sized and large ganglion cells project to the deep stratum griseum superficiale and to the stratum opticum. The ipsilateral projection is restricted to the deep stratum griseum superficiale and stratum opticum and consists predominantly of medium-sized and large ganglion cells.

Monoclonal antibody labels olfactory and visual pathways in Drosophila and Apis brains.

We employed a monoclonal antibody raised against Drosophila brain homogenate for a comparative immunocytochemical analysis of visual and olfactory pathways in brains of two insect species. On Western blots of Drosophila and Apis nervous tissue, antibody fb45 recognized an antigen with an apparent molecular weight higher than 180 kD. Application of the antibody to sections of Drosophila and Apis brain stained certain interneurons which conspicuously fasciculate in common tracts or neuropilar compartments. Both in Drosophila and in Apis, the antigen was also expressed on the perineural sheath and granular cell compartments in the majority of neuronal cell bodies. The antibody stained monopolar cells in the visual system of both species, and in Apis those fibers of the anterior superior optic tract which link the medulla with the mushroom bodies. In Drosophila, bundles of Kenyon cells of the mushroom bodies were stained. In worker bees and drones, the relay neurons of the median and lateral antennoglomerular tracts were labelled. Since the recognition of the antigen does not require fixation, the antibody can be employed to label selectively living neurons in dissociated cell culture. This opens up the possibility for future functional studies on the role of the antigen in vitro.

The sap47 gene of Drosophila melanogaster codes for a novel conserved neuronal protein associated with synaptic terminals.

Proteins expressed specifically in neurons and transported to synaptic terminals are likely to constitute important molecular elements of nervous system function. In an effort to characterize synapse-associated proteins (SAPs) of Drosophila, we have isolated from a hybridoma library several monoclonal antibodies (MABs) that selectively stain synaptic terminals in immunohistochemical preparations. MAB nc46 binds to most but not all synaptic terminals of the Drosophila nervous system, it also recognizes a protein with homologous distribution in other dipteran flies and binds to large parts of fish CNS. In Western blots the antibody labels a Drosophila brain protein of 47 kDa and cross-reacts with brain proteins from several species including insects, fish, mouse and man. From these data we conclude that the corresponding gene has been conserved in evolution at least among diptera. Using MAB nc46 and expression cloning we have identified the 'sap47' gene coding for the 'synapse-associated protein of 47 kDa' of Drosophila melanogaster. Sequence analysis of genomic and cDNA clones reveals the intron-exon structure of the gene and characterizes the complete open reading frames of two alternatively spliced transcripts. The sap47 gene is located in 89A8-B3 on chromosome 3R and codes for two almost identical inferred polypeptides of 347 and 351 amino acids with no significant sequence homology to known proteins.

Inhibition of Entamoeba histolytica cytotoxin by alpha 1 antiprotease and alpha 2 macroglobulin.

We previously reported partial purification of a proteinaceous substance with cytotoxic and enterotoxic activity isolated from the soluble fraction of sonicated axenically cultivated Entamoeba histolytica trophozoites. Demonstration of cytotoxic activity of the preparation (amebal toxin) was dependent on removal of serum from the tissue culture assay system. The objective of the present study was to identify the factor(s) in non-immune sera responsible for producing in vitro inhibition of amebal toxin cytotoxicity on HeLa cells. Gel filtration of non-immune sera from adult humans or bovines demonstrated that two portions of the eluate had significant inhibitory against the toxin. A high molecular weight inhibitory fraction was identified as predominantly alpha-2 macroglobulin and a low molecular weight inhibitory fraction was identified as predominantly alpha-1 antiprotease. Preparative isoelectric focusing of human serum isolated inhibitory fractions containing these same alpha globulins. Alpha-2 macroglobulin was purified and alpha-1 antiprotease was partially purified from human serum by other methods and shown to have high inhibitory activity against the amebal cytotoxin. Substances that were inhibitory to the cytotoxic activity of the amebal toxin also mediated reattachment of toxin treated HeLa cells. We conclude that the characteristics of the serum inhibitors, especially their ability to reverse the cytotoxic effects of amebal toxin on HeLa cells, suggests that the amebal toxin has protease activity.

Residual erythroid progenitors in W/W mice respond to erythropoietin in the absence of steel factor signals.

Erythropoiesis is severely impaired in mice with inactivating mutations in the Steel factor (SF) gene (Sl/Sl mice) or in c-kit, in the SF receptor gene (W/W mice), and in mice with null mutations in the genes for either erythropoietin (EPO) or the erythropoietin receptor (EPO-R). Previous studies indicated that EPO is sufficient for colony development from colony-forming units-erythroid (CFU-E). However, recent studies have shown that there is a physical association between these 2 receptors and that c-kit can phosphorylate EPO-R. To examine the role SF and EPO play in regulating erythropoiesis, we examined the effect of SF and EPO on colony development from cells of the embryonic aorta-gonad-mesonephros (AGM) region, yolk sac, and liver of fetal wild-type and W/W mice. The maturation of CFU-E from these sites did not require the addition of SF to clonal cultures, whereas the efficient development of erythroid bursts required both EPO and SE The number of erythroid colony-forming cells was reduced in both the AGM region and liver of fetal W/W mice. The residual CFU-E present in W/W mice were dependent on EPO and independent of SF. These results indicate that EPO/EPO-R can function to support colony formation in the absence of an SF signal.

Sequential histopathology of cavitary liver abscess. Formation induced by axenically grown Entamoeba histolytica.

Multiple hamster liver passage of Entamoeba histolytica trophozoites with intervening recovery into axenic culture caused increased virulence as measured by increase in the size of the lesion produced. Lesions produced by amebae that had not been liver-passaged did not persist; however, multiply liver-passaged substrains produced large, fluid-filled abscesses one month to six weeks after inoculation. Six days after inoculation, lesions consisted of multiple granulomas, lymphocytes, and E histolytica trophozoites. Large, fluid-filled abscesses produced by liver-passaged substrains lacked the granulomatous appearance of the earlier lesions. The abscesses had a fibrous wall, with E histolytica trophozoites at the inner aspect. To our knowledge, the evolution of early granulomatous lesions into a cavitary abscess with features closely resembling those of human amebic abscess has not been reported previously in the experimental disease in the hamster.

A technique for the electron microscopic autoradiography of Al adenosine receptors in brain tissue. Ligand-receptor fixation by osmium tetroxide.

A procedure for the electron microscopic autoradiography of Al adenosine receptors is described. Fresh tissue slices from rat hippocampus were incubated with the radioactive adenosine analogs: Cyclohexyl[3H]adenosine, 5'-N-ethylcarboxamido[3H]adenosine or or [125I]-iodohydroxyphenylisopropyladenosine. Various fixation agents were tested with respect to the retention of these ligands by the tissue. While most of the ligands were lost in aldehyde fixation they were retained by osmium tetroxide probably via a crosslinking reaction. The final method of choice was an aldehyde prefixation (in the case of [125I]-iodohydroxyphenylisopropyladenosine with 4% buffered paraformaldehyde) during which more than 90% of the nonspecifically bound ligands were washed out while 40% of the specifically bound ligands remained. Subsequent fixation with osmium tetroxide (1%) allowed a standard protocoll for dehydration and embedding to be used with only minimal (less than 5%) further loss of the ligands. Electron microscopic autoradiography provided evidence for a specific distribution of the binding sites for [125I]-iodohydroxyphenylisopropyladenosine.

Synaptic connections of cortical and retinal terminals in the superior colliculus of the rabbit: an electron microscopic double labelling study.

Retinal and cortical afferents to the superior colliculus of the rabbit were labelled simultaneously by injecting 3H-leucine into the right eye and HRP into the left visual cortex. It could be shown that there is some convergence of retinal and cortical input onto common postsynaptic elements in the superficial grey, but these cases were found to be rather rare indicating that most afferents from the retina and the visual cortex terminate either on different postsynaptic cells or on different parts of common postsynaptic cells.

Histamine-like immunoreactivity in the visual system and brain of Drosophila melanogaster.

In this study, immunohistochemistry on cryostat sections is used to demonstrate anti-histamine immunoreactivity in the Drosophila brain. The results support earlier findings that histamine is probably a transmitter of insect photoreceptors. It is further shown that, in Drosophila, all imaginal photoreceptors including receptor type R7 are anti-histamine immunoreactive, whereas the larval photoreceptors do not seem to contain histamine. In addition to the photoreceptors, fibres in the antennal nerve and approximately 12 neurons in each brain hemisphere show strong histamine-like immunoreactivity. These cells arborize extensively in large parts of the central brain.

Cell clones and pattern formation: On the lineage of photoreceptor cells in the compound eye ofDrosophila.

The generalogical relationships of photoreceptor cells within the compound eye ofDrosophila have been studied using cell labelling, with either3H-thymidine or recessive mutations, during the third larval stage. It has been found that photoreceptor and secondary pigment cells arise from different precursor cells. Under the present experimental conditions, precursors of receptor cells give rise to about 8 elements which differentiate as R cells of two different groups. One of the cells differentiates as R7 and the remaining as any one of the R1 to R6. The last cells behave initially as equivalent, and can differentiate within the same or within different, but neighbouring, ommatidia. The class of R1 to R6 cell in which each one of these elements differentiates, seems to depend on the time of its origin. The implications of these findings for the formation of the ommatidial pattern are discussed.

Antibodies to heavy neurofilament subunit detect a subpopulation of damaged ganglion cells in retina.

Neurofilaments ( NFs ) consist of three protein subunits with apparent molecular weights of 68,000 ( 68K ), 145K and 200K , which are found closely associated in most but not all locations in the nervous system. One of these exceptions is the inner retina of the mouse, where antibodies to 145K NFs label large ganglion cells throughout the extent of the cells, while antibodies to 200K NFs label only more distal portions of the optic axons but usually fail to label the ganglion cell somata and proximal axons. Very rarely, however, and more often in old mice, anti- 200K NF antibodies do label a ganglion cell completely. To determine whether these rare, completely labelled cells reflect a pathological alteration, we cut the optic axons, and report here that after a few days some of the axotomized cells could be labelled completely, in a Golgi-like fashion, by anti- 200K NF antibodies. These cells seem to represent the population that forms the projection to the bulk of the lateral geniculate nucleus, as suggested by their size, distribution and projection pattern. Hence, antibodies to the heavy NF subunit in combination with lesions may allow selective retrograde tracing of a subpopulation of ganglion cells, and such antibodies can be used to detect damage in NF-rich neurones at a very early stage, long before they eventually degenerate.

Expression of the Drosophila optomotor-blind gene transcript in neuronal and glial cells of the developing nervous system.

Mutations in the complex gene locus optomotor-blind (omb) can lead to defects in the development of both the optic lobes and external features of the adult fly. We describe here the expression of omb in the developing and adult nervous system using in situ hybridization. During embryogenesis, omb expression is first observed in the optic lobe anlagen. It later expands to a larger part of the developing larval brain and to the gnathal lobes. Cells in the ventral and peripheral nervous systems begin to express omb after completion of germ band extension. Later in embryonic development, expression declines and only persists in the antennomaxillary complex and in part of the brain hemispheres. During the larval and pupal stages, omb expression in the brain is confined to the developing optic lobes and contiguous regions of the central brain. At these stages, only a few cells show expression in the ventral ganglion. In the eye imaginal disc, transcript accumulation is most conspicuous in a group of presumptive glia precursor cells posterior to the morphogenetic furrow and in the optic stalk. In the adult brain, expression is prominent in several regions of the optic lobe cortex and along the border between central brain and optic lobes. In the mutation In(1)ombH31, 40 kb of regulatory DNA, downstream from the transcription unit, are removed from the omb gene. In(1)ombH31 is characterized by the lack of a set of giant interneurons from the lobula plate of the adult optic lobes. We find that, already during embryogenesis, there is a drastic difference between wild type and In(1)ombH31 in the level of the omb transcript in the optic lobe primordia. The adult mutant phenotype may thus be caused by omb misexpression during embryonic development.

Oxaceprol, an atypical inhibitor of inflammation, reduces leukocyte adherence in mouse antigen-induced arthritis.

Oxaceprol (N-acetyl-L-hydroxyproline), an atypical inhibitor of inflammation, is an established drug forjoint disease without serious side-effects. Recent studies have emphasized that oxaceprol has an effect on the microcirculation. Since the exact mechanism of action remains unclear, the aim of our study was to investigate the leukocyte-endothelial cell interactions in oxaceprol-treated mice with antigen-induced arthritis (AiA) using intravital microscopy. In our study, Balb/c mice were allocated to 4 groups (n 7, 8, 8, 8): 2 control groups with saline or oxaceprol and 2 groups of arthritic animals which received saline or oxaceprol (100 mg/kg twice a day intraperitoneally). The severity of arthritis was quantified by the transverse knee joint diameter. For the intravital fluorescence microscopy measurements on day 10 after inducing arthritis, the patella tendon was partily resected to visualize the intraarticular synovial tissue of the knee joint. The number of rolling and adherent leukocytes as well as RBC velocity and functional capillary density (FCD) were quantified in synovial microvessels. Furthermore, leukocyte infiltration was determined in the histological sections with an established score. No significant changes in mean arterial blood pressure or functional capillary density were found in any of the groups. However, the leukocyte rolling fraction and number of leukocytes adherent to the endothelium were increased in postcapillary venules of the synovium in arthritic animals (0.16 to 0.31, 78 cells/mm2 to 220 cells/mm2). In animals with AiA treated with oxaceprol, leukocyte adherence and swelling were significantly reduced in comparison to the arthritic animals treated with saline. Furthermore, the histological score showed less leukocyte infiltration in the oxaceprol treated arthritic animals. Thus, oxaceprol reduces leukocyte adherence in vivo and leukocyte infiltration in mouse AiA, indicating an effect on synovial microcirculation.

Entamoeba histolytica: purification of cathepsin B.

A cytotoxic cysteine proteinase with a molecular weight of 16,000 was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified from frozen-thawed strain HM-1 by ion-exchange chromatography on DEAE-cellulose, organomercurial agarose affinity chromatography, and size-exclusion chromatography. The purified enzyme had proteinase activity that could be demonstrated on azocasein (pH 5), hemoglobin (pH 5), or carbobenzoxy-L-arginyl--L-arginyl-7-amino-4-trifluoromethylcoumarin++ + (Z-arg-arg-AFC), a substrate specific for cathepsin B. Enzyme activity was stable to high pH, but not to 40 C for 1 hr or 56 C for 0.5 hr. As typical of cysteine proteinases, inhibition of activity on Z-arg-arg-AFC by p-chloromercuribenzoate or mercury was reversed by free sulfhydryl groups. Both the proteinase and cytotoxic activities of the purified amoebal cathepsin B were inhibited by leupeptin and serum and activated by free sulfhydryl groups, supporting the hypothesis that both activities are characteristics of amoebal cathepsin B. Virulent strains of E. histolytica (HM-1 and Rahman) had significantly more cathepsin B activity per milligram protein than less virulent strains (HK-9, Laredo, and Huff). The correlation between higher levels of cathepsin B activity in strains with greater virulence could indicate a role for amoebal cathepsin B in the pathogenesis of amoebiasis.