J. Bogers

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract.

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.

Schistosoma mansoni infection causing diffuse enteric inflammation and damage of the enteric nervous system in the mouse small intestine.

Schistosomiasis mansoni is a major health problem, mainly occurring in developing countries. A large proportion of infected individuals suffers from motility‐related gastrointestinal problems. In the present study, the diffuse inflammatory response in the small bowel wall, as compared to the egg‐induced granulomatous inflammation, was investigated. For this purpose, OF1 mice infected with Schistosoma mansoni 8–16 weeks prior to the experiment, and uninfected control mice were studied. The ileum showed both a diffuse mucosal inflammation as well as a granulomatous reaction. The diffuse mucosal inflammation caused an increase in the thickness of the mucosa, with blunting of the villi. A significant, transient increase of thickness of the muscularis propria after 12 weeks of infection was noted. There was an infection‐related mast cell infiltrate in the muscularis propria, consisting of formalin fixation‐insensitive connective tissue mast cells. Ganglionitis of the myenteric plexus was noted. Rarely, ganglia of the myenteric plexus contained apoptotic cells. A general pharmacological set of experiments showed a significant increase in intestinal contractility, both to exogenously administered, as well as to endogenously released neurotransmitters. Our results demonstrate that S. mansoni infection in the mouse ileum leads to diffuse specific enteric inflammation that is associated with an enhanced response to contractile agents.

Histomorphometric Study of the Normal Middle Ear Mucosa: Preliminary Results Supporting the Gas-Exchange Function in the Postero-Superior Part of the Middle Ear Cleft

This study concerns the distance between the centre of gravity of blood vessels and the basement membrane of the middle ear cleft mucosa. The measurements were performed perpendicular to the long axis of the cross-section of the vessels, and revealed a significant difference between two regions of the middle ear cleft. At the level of the postero-superior part (epitympanum, aditus ad antrum, mastoid antrum and highest part of the mastoid air cells system), the distance between the blood vessels and the basement membrane of the mucosa was statistically shorter than in the antero-inferior part of the middle ear cleft. This indicates a privileged function of gaseous exchange of the postero-superior part of the middle ear cleft and may divide the middle ear cleft into different functional parts.

Thin-layer liquid-based cervical cytology and PCR for detecting and typing human papillomavirus DNA in Flemish women

The objective of this study was to document the occurrence and to correlate the prevalence of different human papillomavirus (HPV) types with the cytological results on simultaneously performed thin-layer preparations in a large population of Flemish women. During 1 year, 69 290 thin-layer preparations were interpreted using the Bethesda classification system. Using an algorithm for HPV testing based on consensus primers and type-specific PCRs in combination with liquid-based cytology, we determined the occurrence and distribution of 14 different oncogenic HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Reflex HPV testing was performed on cytologically abnormal samples and on an age matched randomly selected control group with normal cervical cytology (n=1351). Correlation between cytology, age and prevalence for the 14 different high-risk HPV types is given. There is a significant increase in predominance of high-risk HPV types, with increasing abnormal cytology. Coinfection with multiple HPV types also increased with cytological abnormalities, and was highest in HSIL (16.7%). In Flanders, HSIL was most often associated with HPV types 16, 33, 35, 31, 18 and 51. Using thin-layer liquid-based cytology and PCR to detect HPV, it is feasible to screen large numbers of women.

Ki-67 immunocytochemistry in liquid based cervical cytology: useful as an adjunctive tool?

Aims: To test the ability of Ki-67 to detect cytological lesions in a screening setting and its use as a surrogate marker of human papillomavirus (HPV) infection. Methods: A study of liquid based cytology, HPV DNA testing by MY09/MY11 consensus polymerase chain reaction (PCR), type specific PCRs, and Ki-67 immunocytochemistry on a randomly selected series of 147 patients. Results: Comparison of the number of Ki-67 immunoreactive cells/1000 cells in the different cytological groups showed that the HSIL group yielded a significantly higher mean count than did the other groups. The number of Ki-67 immunoreactive cells/1000 cells was significantly higher in HPV-16 positive samples than in samples containing infections with other high risk types. Receiver operating characteristic curves indicated a test accuracy (area under curve) of 0.68, 0.72, and 0.86 for atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesions (LSIL), and high grade squamous intraepithelial lesions (HSIL), respectively. Thresholds for 95% sensitivity were 0.07, 0.08, and 0.15 Ki-67 immunopositive cells/1000 cells for ASCUS, LSIL and HSIL, respectively. The threshold for 95% specificity was 1.9 Ki-67 immunopositive cells/1000 cells. Conclusions: Ki-67 immunocytochemistry can be applied to liquid based cytology. The accuracy and diagnostic indices of the test are good when compared with those of other techniques. As part of a panel of screening procedures, it could be used as an adjunct to liquid based cytology to identify HSIL, and as a surrogate marker of HPV-16 infection.

Immunofluorescent Visualization of the Excretory and Gut System of Schistosoma mansoni by Confocal Laser Scanning Microscopy

Conventional epifluorescence microscopy (CEM) and confocal laser scanning microscopy (CLSM) were used to visualize the excretory system and the gut on whole organisms of different life-cycle stages of Schistosoma mansoni. To visualize the gut system, an anti-circulating anodic antigen (CAA) monoclonal antibody (MAb) (120-1B10-A) was used, whereas the excretory system was immunohistochemically stained with an anti-flame cell MAb (51-4H8-A) and with a recently described anti-egg MAb (114-5B1-A). The CEM procedure resulted in clear images at low magnification but the signal-to-noise ratio on the higher magnification images was very poor. Using CLSM on the adult worm, the 114-581-A MAb demonstrated a well-defined system of canals that could be morphologically identified as the excretory system. The flame cells terminating the branches of the excretory canals showed a clear immunoreactivity with the 114-5B1-A MAb as well as with the specific flame cell MAb. The gut system could be visualized, using an anti-CAA MAb, as two well-defined bands throughout the length of the parasite. Application of the 114-5B1-A MAb on cercariae revealed a strong fluorescence on the cercarial surface, whereas no immunoreactivity could be detected on internal structures. Whole eggs showed a bright fluorescence of the egg shell, whereas miracidia showed immunoreactivity of the germinal cells located in the center of the organism. The CLSM procedure, especially with the recently introduced fast photon-counting option, provides a superior tool to investigate the three-dimensional localization of different epitopes on immunofluorescently stained whole mounts of multicellular organisms in comparison with CEM.(ABSTRACT TRUNCATED AT 250 WORDS)

Helicobacter pylori, parietal cell antibodies and autoimmune gastropathy in type 1 diabetes mellitus

Fifteen to 20% of type 1 diabetic patients exhibit parietal cell antibodies (PCA), which are associated with autoimmune gastritis, hypochlorhydria, iron deficiency and pernicious anaemia.

Role of reactive nitrogen species in neuronal cell damage during intestinal schistosomiasis

Abstract. Free radicals are known to be involved in the host reaction during Schistosoma mansoni-induced inflammation in the liver and the intestine. In the present study, the influence of reactive nitrogen species (RNS) on the enteric neurons of infected ileum of mice was investigated. Cryosections and whole-mounts of the ileum of control, and 8- and 15-week-infected mice were processed for immunohistochemical localization of 3-nitrotyrosine, a biomarker of RNS, and of active caspase-3, a key executioner of apoptosis. An antibody directed against protein gene product 9.5 or S100 protein was used as a marker for neurons or enteroglial cells. In infected mice, but not in control animals, 3-nitrotyrosine was detected in parasite eggs and, as revealed by double immunolabelling, in some neuronal and enteroglial cells. Quantitative analysis of whole-mounts showed that the percentage of 3-nitrotyrosine-immunoreactive neurons significantly increased with time in both the submucous and myenteric plexus. Caspase-3 immunoreactivity was predominantly found in parasite eggs in infected mice. Immunoreactive enteric neurons were occasionally observed. The results indicate that inflammation-induced RNS are present in the ileum of S. mansoni-infected mice, and participate in the elimination of the schistosome eggs causing damage in a significant number of enteric neurons. However, neuronal cell death appears to be a rare phenomenon in the schistosome-infected mouse ileum.

Effects of fixation on RNA integrity in a liquid-based cervical cytology setting

Aims: Despite many improvements, cervical cancer screening is still subject to shortcomings. Diagnostic accuracy may improve by using molecular biological techniques, requiring RNA of superior quality. This study determined the effect of SurePath fixation on RNA integrity to assess the suitability of clinical samples collected in this medium for RNA-based molecular assays. Methods: RNA isolation was performed on fresh and fixed HeLa cells and exfoliated cervical cells fixed in SurePath. The RNA integrity was evaluated by analysis of ribosomal RNA as an indicator of quality. The effect of SurePath preservation on PCR amplification was evaluated by real-time reverse transcriptase (RT)-PCR. Results: In contrast to unfixed cells, SurePath-fixed cells yielded less and severely degraded RNA, as shown by the absence of ribosomal RNA bands. RNA derived from SurePath-fixed cells showed poor real-time RT-PCR amplification characteristics, as evidenced by the absent correlation between threshold values and log cDNA concentration. Conclusions: Implementation of molecular biology in a clinical context is on the rise and may alleviate shortcomings in current screening and diagnostics. This study shows that SurePath fixation gives rise to highly fragmented RNA with insufficient quality for further reliable analysis by standard real-time RT-PCR applications. The increasing prominence of molecular screening stresses the importance of this finding, which must be considered in relation to choice of an appropriate liquid-based cytology system.

Quantitative and morphological aspects of Unicryl versus Lowicryl K4M embedding in immunoelectron microscopic studies.

In this study we compared the recently commercialized electron microscopy embedding resin Unicryl with the well-known resin Lowicryl K4M with regard to morphological and immunohistochemical preservation properties. The standard embedding procedure recommended by the manufacturer for the use of Unicryl resulted in considerable morphological alterations of the tissue, with the appearance of large gaps in and between the cells of the examined tissue. Morphometric analysis pointed to a swelling of the extracellular matrix as the main cause of these morphological artifacts. A slight modification in the protocol to correct this artifact is proposed and tested. Immunohistochemically, tissue embedded in Unicryl resulted in a significantly stronger immunogold labeling than identical tissue embedded in Lowicryl K4M. From the results of this technical study, it can be concluded that Unicryl embedding is a valuable new tool to supplement the available techniques for immunoelectron microscopic studies.