Anterograde tracing and immunohistochemical characterization of potentially mechanosensitive vagal afferents in the esophagus.

Vagal mechanosensitive afferents with an important functional role in esophageal peristalsis are well known from physiological studies. It is not known whether these fibers represent a separate subpopulation among all vagal afferents projecting to the esophageal wall. A morphological and immunohistochemical description of vagal afferents was undertaken to define their possible homo- or heterogeneity. The peripheral projections of vagal afferents were anterogradely labeled by injection of wheatgerm agglutinin conjugated to horseradish peroxidase into the nodose ganglion of rats. The anterogradely transported tracer was detected by tyramide amplification in conjunction with immunohistochemistry for Ca(2+)-binding proteins recently identified in different types of mechanosensory endings. It was found that vagal afferents represented a morphologically and structurally homogeneous population projecting to the myenteric ganglia of the esophagus, where they terminated as highly branched endings. Vagal afferent terminals, however, were different in their staining intensity for calretinin and calbindin, which ranged from intense to no detectable immunofluorescence. The fluorescence intensity of Ca(2+)-binding proteins within the vagal terminating branches was graded and the average staining intensity determined of all terminating branches in the upper, middle, and lower thirds of the esophagus. The average staining intensity was highest in the upper third of the esophagus and then declined in a statistically significant manner in the middle and lower thirds. This result suggests different requirements for intracellular Ca(2+)-buffering capacities in vagal afferents depending on their position along the esophageal axis and corroborates studies reporting a segmental organization of esophageal motility. Immunohistochemical evidence of substance P (SP) in a subset of vagal terminals was demonstrated. Hence, an effector role of vagal afferents on esophageal peristalsis by the release of SP, as has been proposed by physiological studies, is also supported by immunohistochemical data.

Novel alternatively spliced isoforms of the neurofibromatosis type 2 tumor suppressor are targeted to the nucleus and cytoplasmic granules.

We cloned novel splice variants Mer150, Mer151 and Mer162 of the neurofibromatosis 2 (NF2) tumor suppressor, which demonstrate a tissue-specific and development-specific expression pattern. Isoform Mer150 is created by cryptic splicing from exon 8 to 14 and represents an N-terminal truncation of 259 residues. Mer151 is characterized by in-frame splicing out of several exons and a modified C-terminus due to a frameshift in exons 13+14 and premature termination. Mer162 represents a head-to-tail isoform resulting from in-frame skipping of exons 5-16. As a common feature, the alpha-helical domain and a variable proportion of the ERM homology domain are spliced out in these isoforms. To investigate differences in subcellular localization, we expressed epitope-tagged cDNA constructs of the wild-type NF2 as well as of the three alternatively spliced transcripts in NIH 3T3 cells by nuclear microinjection or lipid-mediated transfection. Subcellular localization of Mer151 in filopodia and ruffling membranes was similar to the wild-type NF2. Mer151, however, was targeted to the nucleus, which was not observed for wild-type NF2, Mer150 or Mer162. A putative nuclear localization signal created by alternative splicing was identified in Mer151. In contrast to Mer151, Mer150 and Mer162 were not found in regions of the plasma membrane, but localized to a granular intracellular compartment. The results suggest that the recently described actin-binding domain in exon 10, but not the presence or absence of exons 2+3, is relevant for subcellular targeting. Although the NF2 protein is known as a cytoskeletal linker, additional functions in a cytoplasmic compartment and in the nucleus may exist.

[SPIO-enhanced MR angiography for the detection of venous thrombi in an animal model]. / SPIO-unterstützte MR-Angiographie zur Detektion venöser Thromben im Tiermodell.

PURPOSE: An animal model is used to investigate whether MR angiography combined with super-paramagnetic particles of iron oxide (SPIO) is suitable for detecting thromboses. METHODS: 42 rats in groups of 7 each were examined on days 1, 3, 5, 7, 9 and 11, respectively, after mechanical/chemical thrombus induction in a 1.5 Tesla magnet with a FISP sequence (TR/TE/FA 50 ms/6 ms/40 degrees). Imaging was performed before and up to 90 minutes after intravenous injection of 30 mumol FE/kg BW of the experimental SPIO (hydrodynamic diameter, 34 +/- 17 nm LLS; R1 and R2 relaxivity at 0.47 T, 31 and 57 L/(mmol*s)). MIP reconstructions of MR angiographies were submitted to consensus assessment by two examiners. using histology as the gold standard. RESULTS: The image quality of MIP reconstructions was rated as good in 38 of 42 cases. With regard to thrombotic vessel occlusion, MR angiography coincided with histology in 17 of 42 cases and differed in 25, lumen narrowing being overestimated by MRI in 4 cases and underestimated in 21. In these 21 cases, the histologically observed recanalization could not be detected by MR angiography. CONCLUSIONS: SPIO-enhanced MR angiography achieves adequate vessel contrast and reliable thrombus detection in animal experiments. Partial volume effects prevent microscopically observed recanalization from being detected by clinical MRI.

Vagal efferent and afferent innervation of the rat esophagus as demonstrated by anterograde DiI and DiA tracing: focus on myenteric ganglia.

Anterograde tracing with the carbocyanine tracer DiI and the aminostyrol derivative DiA was used to selectively label fibers from the nucleus ambiguus, dorsal motor nucleus and nodose ganglion, respectively, terminating in the rat esophagus, and to compare them with the innervation of the gastric fundus in the same animals. Ambiguus neurons terminated on motor endplates distributed mainly to the ipsilateral half of the esophagus. There was no evidence of preganglionic innervation of myenteric ganglia from ambiguus neurons. Neurons of the dorsal motor nucleus supplied sparse fibers to only about 10% of enteric ganglia in the esophagus while they innervated up to 100% of myenteric ganglia in the stomach. Neurons of the nodose ganglion terminated profusely on more than 90% of myenteric ganglia of the esophagus and on about 50% of ganglia in the stomach. Afferent vagal fibers were also frequently found in smooth muscle layers starting at the esophago-gastric junction. In contrast, they were extremely rare in the striated muscle part of the esophagus. These morphological data suggest a minor influence of neurons of the dorsal motor nucleus and a prominent influence of vagal afferent terminals onto myenteric neurons in the rat esophagus.

Tyramide amplification allows anterograde tracing by horseradish peroxidase-conjugated lectins in conjunction with simultaneous immunohistochemistry.

Current protocols for a combined approach of anterograde tracing with carbocyanine dyes or horseradish peroxidase (HRP) conjugates and immunohistochemistry represent a compromise between sensitive detection of the tracer and the immunohistochemical procedure. Therefore, it was investigated whether the use of tyramide amplification allows sensitive anterograde tracing with wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) in conjunction with simultaneous immunohistochemistry. Vagal afferents were anterogradely labeled by injection of WGA-HRP into the nodose ganglion of rats. By use of tyramide-biotin amplification, a dense fiber plexus of vagal afferents was visualized centrally in the nucleus of the solitary tract and in retrogradely labeled neurons in the dorsal vagal nucleus. In the esophagus and duodenum, large- and small-caliber vagal fibers and terminals could be demonstrated comparably to conventional tracing technique using carbocyanine dyes or WGA-HRP and TMB histochemistry. Combination with immunohistochemistry could easily be done, requiring only one more incubation step, and did not result in loss of sensitivity of the tracing. With this method and confocal microscopy, the presence of Ca binding proteins in vagal afferent terminals could be demonstrated. Tyramide amplification allows sensitive anterograde tracing with low background staining in conjunction with immunohistochemistry of a-axonal markers.

Subcellular localization and expression pattern of the neurofibromatosis type 2 protein merlin/schwannomin.

To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.

Therapy with CD7 monoclonal antibody TH-69 is highly effective for xenografted human T-cell ALL.

Human T-cell acute lymphocytic leukaemia (ALL) was established in athymic nude or severe combined immunodeficient (SCID) mice by injecting CEM or MOLT-16 cells. When nude mice bearing approx. 2 g of tumour were treated with a single injection of CD7 antibody TH-69, 82.6% reached complete remission within 10 d whereas 13.0% showed partial remission. Similarly, in SCID mice with advanced disease a significant prolongation of survival was seen. The therapeutic effects were dependent upon dose and affinity of the antibody. TH-69 is a high-affinity antibody (7.6 x 10(9) M-1) that rapidly induced modulation during treatment. The Fc-portion of the antibody was required for effective tumour cell killing. Complement deposition was found on tumour sections after TH-69 treatment and in part may account for tumour destruction. There was no evidence for antibody-dependent cellular cytotoxicity (ADCC). The kinetics of tumour disappearance suggested the initiation of a programmed cell death (PCD), despite the lack of significant DNA fragmentation. Unmodified high-affinity antibodies to the T-cell antigen CD7 have potential for T-cell ALL therapy.

Vagal sensors in the rat duodenal mucosa: distribution and structure as revealed by in vivo DiI-tracing.

Results from functional studies point to the importance of chemoreceptive endings in the duodenum innervated by vagal afferents in the regulation of gastrointestinal functions such as gastric emptying and acid secretion, as well as in the process of satiation. In order to visualize the vagal sensory innervation of this gut segment, vagal afferents were selectively labeled in vivo by injecting the lipophilic carbocyanine dye DiI into either the left or the right nodose ganglion of young adult rats. Thick cryostat sections or whole-mounted peels of muscularis externa or submucosa of formalin-fixed tissue were analyzed with conventional and/or confocal microscopy. In the mucosa, many DiI-labeled vagal afferent fibers were found with terminal arborizations mainly between the crypts and the villous lamina propria. In both areas, vagal terminal branches came in close contact with the basal lamina, but did not appear to penetrate it so as to make direct contact with epithelial cells. Labeled vagal afferent fibers in the villous and cryptic lamina propria were found to be in intimate anatomical contact with fibrocyte-like cells that may belong to the class of interstitial cells of Cajal, and with small granular cells that might be granulocytes or histiocytes. Although our analysis was not quantitative, and considering that labeling was unilateral and not complete, it appears that the overall density of vagal afferent mucosal innervation was variable; many villi showed no evidence for innervation while other areas had quite dense networks of arborizing terminal fibers in several neighboring villi. Analysis of separate whole-mounted muscularis externa and submucosa peels revealed the presence of large bundles of labeled afferent fibers running within the myenteric plexus along the mesenteric attachment primarily in an aboral direction, with individual fibers turning towards the antimesenteric pole, and either penetrating into the submucosa or forming the characteristic intraganglionic laminar endings (IGLEs). Although the possibility of individual fibers issuing collaterals to myenteric IGLEs and at the same time to mucosal terminals was not demonstrated, it cannot be ruled out. These anatomical findings are discussed in the context of absorptive mechanisms for the different macronutrients and the implication of enteroendocrine cells such as CCK-containing cells that may function as intestinal "taste cells".

Vagal afferent innervation of rat abdominal paraganglia as revealed by anterograde DiI-tracing and confocal microscopy.

Abdominal vagal afferent fibers were selectively labeled by injecting the fluorescent carbocyanine dye DiI into the left nodose ganglion of rats. Almost all paraganglia that were distributed along the five major abdominal vagal branches and their subbranches were found to be innervated by labeled vagal afferents. Laser scanning confocal microscopy with its single optical sectioning and three-dimensional reconstruction capabilities were used to analyze this innervation in more detail for paraganglia near the vagal hepatic branch and liver hilus. Furthermore, in double-labeling studies, it was demonstrated that a large percentage of the vagally innervated glomus cells were capable of catecholamine synthesis on the basis of their positive staining for tyrosine hydroxylase antibody. These findings support the concept of a chemoreceptive function for the abdominal paraganglia.

Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA.

The occurrence and spatial distribution of intracellular DNA fragmentation was investigated by in situ 3' end labelling of DNA breaks in K562 cells treated in such a way to cause either apoptotic or necrotic cell death. The localisation of DNA breaks was examined by confocal laser microscopy and compared with the electron-microscopic appearance of the cells. In addition, the number of cells with fragmented DNA was counted and compared with the number of dead cells, as determined by the nigrosin dye exclusion test. Apoptosis was induced by cultivation of the cells in the presence of actinomycin D. Cells undergoing apoptosis were characterised by massive intracellular DNA fragmentation that was highly ordered into successive steps. Cells in early stages of the apoptotic process had DNA breaks diffusely distributed in the entire nucleus, except the nucleolus, with crescent-like accumulations beyond the nuclear membrane. In the more advanced stages, the nucleus was transformed into many round bodies with intense labelling. Intracellular accumulations of fragmented DNA corresponded exactly to electron-dense chromatin seen in the electron microscope, whereas diffuse DNA breaks had no morphological correlate at the ultrastructural level. In necrosis induced by ionomycin, NaN3, or rapid freezing combined with thawing, no DNA fragmentation occurred at the onset of cell death, but appeared 24 h later. This fragmentation was not characterised by a unique morphology, but represented the breakdown of the chromatin in the configuration remaining after cell death. Therefore, apoptosis is characterised by DNA fragmentation that proceeds in a regular orderly sequence at the beginning of cell death, and can be detected by in situ 3'end labelling of DNA breaks.

Borrelia burgdorferi-induced ultrastructural alterations in human phagocytes: a clue to pathogenicity?

A chronic infection with the spirochaete Borrelia burgdorferi typically results in a multistage, multisystem illness. Thus, Lyme borreliosis may provide an interesting model to study the pathomechanisms of microbial persistence. In the present investigation, human peripheral blood monocytes, polymorphonuclear leukocytes, and synovial macrophages were incubated with B. burgdorferi and examined by light and electron microscopy. It was found that incubation with the spirochaetes induced distinct features in the phagocytes. Features which may be related to the pathogenesis of Lyme disease included the segmental uptake of spirochaetes with leaky lysosomes, the invagination of large membrane areas, the extra-lysosomal degradation of internalized B. burgdorferi cells and, finally, the formation of mononuclear syncytial cells and homotypic cell clusters. Features of unknown relevance were the occurrence of two types of cytoplasmic inclusion bodies and exocytic vesicles. These novel findings suggest that reactive alterations of the phagocytes may contribute to the pathogenesis of Lyme borreliosis, which could help to focus future histopathological studies. Moreover, these results may provide new insights into the pathogenesis of other infectious diseases characterized similarly by microbial persistence.

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy.

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.

An anterograde tracing study of the vagal innervation of rat liver, portal vein and biliary system.

In order to investigate the distribution and structure of the vagal liver innervation, abdominal vagal afferents and efferents were selectively labeled by injecting WGA-HRP or Dil into the nodose ganglia, and DiA into the dorsal motor nucleus, respectively. Vagal afferent fibers produced characteristic terminal-like structures at three locations in the liver hilus: 1. Fine varicose endings preferentially surrounding, but not entering, the numerous peribiliary glands in the larger intra and extrahepatic bile ducts 2. Large, cup-shaped terminals in almost all paraganglia 3. Fine varicose endings in the portal vein adventitia. No fibers and terminals were found in the hepatic parenchyma. While about two thirds of the vagal afferent fibers that originate in the left nodose ganglion, and are contained in the hepatic branch, bypass the liver hilus area on their way to the gastroduodenal artery, a significant number (approx. 10% of the total) of vagal afferents that do innervate the area, originates from the right nodose ganglion, and projects to the periarterial plexus of the common hepatic artery and liver pedicle most likely through the dorsal celiac branch. Varicose vagal efferent fibers were present within the fascicles of the vagal hepatic branch and fine terminal-like structures in a small fraction of the paraganglia. No efferents were found to terminate in the hepatic parenchyma or on the few neurons embedded in nerves or paraganglia. In contrast to the paucity of vagal terminals in the hepatic parenchyma, an abundance of vagal efferent and afferent fibers and terminals with distinctive distribution patterns and structural characteristics was present in esophagus and gastrointestinal tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Myelofibrosis in primary myelodysplastic syndromes: a retrospective study of 352 patients.

In a retrospective study of 352 patients with primary myelodysplastic syndromes, 61 (17.3%) revealed myelofibrosis in bone marrow biopsies. The fibrosis was observed to occur mostly focally (41/61 cases), and collagen deposits were found very rarely (4/61). The histopathology of bone marrow biopsies revealed hyperplasia and disturbed differentiation in megakaryopoiesis; the frequency and grade of dysplasia in megakaryopoiesis increased with advancing myelofibrosis. Reticulin fibrosis occurred in all subtypes of MDS; however, there was a higher incidence in chronic myelo-monocytic leukaemia (CMMoL). The frequency of cytogenetic aberrations was significantly higher in the MDS cases with myelofibrosis, compared to the cases without fibrosis. Clinical data showed significantly lower values of haemoglobin and lower platelet counts in MDS with myelofibrosis. Life expectancy was reduced to 9.6 months, compared with 17.4 months in MDS without fibrosis. In refractory anaemia, the survival times were 10.0 months in MDS with myelofibrosis, compared to 28.9 months in MDS without myelofibrosis. 36.6% of the patients with MDS and myelofibrosis developed a transformation into ANLL during the course of the disease. Myelofibrosis therefore seems to herald a poor prognosis.