Cytokeratin-positive meningeal peripheral PNET/Ewing's sarcoma of the cervical spinal cord: diagnostic value of genetic analysis

Peripheral primitive neuroectodermal tumor (PNET)/Ewing's sarcoma (ES) of the central nervous system is extremely rare and should be differentiated from central PNET and other small blue round cell tumors. We describe a case of a meningeal peripheral PNET/ES of the spinal cord in an 11-year-old boy. Immunohistochemically, the small blue round cell tumor showed expression of epithelial markers and of CD99, thus posing an important differential diagnostic problem with a poorly differentiated synovial sarcoma. Fluorescence in situ hybridization revealed rearrangement of the EWS gene, as seen in peripheral PNET/ES. Peripheral PNET/ES does occur in the central nervous system, but its diagnosis can be extremely difficult on morphologic and immunohistochemical grounds alone. Genetic analysis plays a key role in its distinction from other small blue round cell tumors.     

Barrett's esophagus is characterized by expression of gastric-type mucins (MUC5AC,MUC6) and TFF peptides (TFF1 and TFF2), but the risk of carcinoma development may be indicated by the intestinal-type mucin,MUC2

Barrett's esophagus (BE) consists of metaplastic epithelium of the esophagus, generally diagnosed by mucin histochemistry. We aimed to determine which mucins were expressed in BE, and to relate their expression to BE pathology. Archival biopsies of 4 patient groups were selected, based on standard histochemistry: BE without inflammation, BE with inflammation, ulcerating BE, and BE with dysplasia. Sections were stained by immunohistochemistry for secretory mucins (MUC2, MUC5AC, MUC5B, and MUC6), the proliferation marker Ki-67, and mucin-associated trefoil factor family (TFF) peptides (TFF1, TFF2, and TFF3). MUC5AC and TFF2 were expressed at similar high levels in each clinical group. Intestinal metaplasia (IM), detected both histochemically and by the intestinal mucin MUC2, was lowest in inflamed BE. The expression of the intestinal-type TFF3 did not differ among the groups. Ulcerating BE was distinguished by very low expression of MUC6 and MUC5B, but very high expression of TFF1. Proliferation was not different among the groups. In the total group of BE patients, H. pylori infection of the stomach correlated with decreased TFF2 expression in the BE epithelium. We conclude that BE is best characterized by the specific expression of the gastric-type markers, MUC5AC, MUC6, TFF1, and TFF2. Ulcerating BE constitutes the most distinguished group with respect to mucin and TFF expression. Of the intestinal markers, MUC2 is very specific for IM in BE, whereas TFF3 is not a marker for IM. The low occurrence of IM in inflamed BE suggests that these patients may have the lowest risk of developing carcinoma.     

Evaluation of cost functions for gray value matching of two-dimensional images in radiotherapy

In external beam radiotherapy, portal imaging is applied for verification of the patient setup. Current automatic methods for portal image registration, which are often based on segmentation of anatomical structures, are especially successful for images of the pelvic region. For portal images of more complicated anatomical structures, e.g., lung, these techniques are less successful. It is desirable to have a method for image registration that is applicable for a wide range of treatment sites. In this study, a registration method for two-dimensional (2D) registration of portal and reference images based on intensity values was tested on portal images of various anatomical sites. Tests were performed with and without preprocessing (unsharp mask filtering followed by histogram equalization) for 96 image pairs and six cost functions. The images were obtained from treatments of the rectum, salivary gland, brain, prostate, and lung. To get insight into the behavior of the various cost functions, cost function values were computed for each portal image for 20,000 transformations of the corresponding reference image, translating the reference image in a range of +/- 1 cm and rotating +/- 10 degrees with respect to the clinical match. The automatic match was defined as the transformation associated with the global minimum (found by an exhaustive search). Without preprocessing, the registration reliability was low (less than 27%). With preprocessing, about 90% of the matches were successful, with a difference with our gold standard (manual registration) of about 1 mm and 1 degree SD. All tested cost functions performed similarly. However, the number of local minima using mutual information was larger than for the other tested cost functions. A cost function based on the mean product of the corresponding pixel values had the least number of local minima. In conclusion, gray value based registration of portal images is applicable for a wide range of treatment sites. However, pre-processing of the images is essential.     

Selective developmental defects of cord blood antigen-presenting cell subsets

Defective antigen-presenting cell (APC) function has been hypothesized to contribute to increased infection susceptibility in newborns. We used multiparameter flow cytometry to characterize APC subsets in adult peripheral blood (APB) and cord blood (CB). APB had a higher proportion of CD11c+ dendritic cells (DC), whereas CB mainly contained CD123+ DC. APB was enriched in CD16+CD11c+ DC subset, whereas CD34+CD11c-CD123lo cells were prominent in CB. Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production was dampened in myeloid DC and monocytes from CB, whereas IL-1alpha production was not different. The reduction in TNF-alpha response did not appear to result from reduced surface detection of LPS, because CD14, toll-like receptor (TLR)-4 and TLR-2 levels were not reduced in CB APC compared with APB cells. Also, there was no correlation between TLR-2 or TLR-4 levels and TNF-alpha production in myeloid DC and monocytes. CB monocytes had lower surface HLA-DR immediately ex vivo. Both APB and CB monocytes upregulated HLA-DR after incubation, but an additional LPS-induced increase in HLA-DR was suggested only in APB monocytes. APB monocytes also showed a greater LPS-induced increase in CD40 expression. Together, our data show significant, selective differences in circulating APC between neonates and adults.     

Adsorption of fibronectin derived from serum and from human endothelial cells onto tissue culture polystyrene

Human endothelial cells (HEC) suspended in a culture medium containing 20% human serum (CMS) adhere and spread on(to) moderately wettable polymers, such as tissue culture polystyrene (TCPS). We have previously shown that serum derived-fibronectin, which is a cell adhesion promoting protein, has a high affinity for TCPS, but that the amount of fibronectin which adsorbed from CMS was relatively small. In this study we investigated whether fibronectin derived from HEC contributes to the adhesion and spreading of the cells on(to) TCPS. Therefore, HEC were seeded in the presence of fibronectin-depleted CMS. The amount of fibronectin detected on TCPS increased with both cell seeding density and incubation time. Although initial HEC adhesion is delayed on TCPS which has been precoated with albumin (Alb), high density lipoprotein (HDL) or immunoglobulin G (IgG), maximal numbers of adhering and spreading HEC were found on these surfaces 6 h after seeding of HEC. Fibronectin was detected on these surfaces, but an exchange of preadsorbed Alb, HDL, or IgG for fibronectin could not be demonstrated. We conclude that HEC deposit fibronectin onto TCPS, irrespective of the presence of a preadsorbed layer of proteins which delay cell adhesion.     

Metaplasia of the duodenum shows a Helicobacter pylori-correlated differentiation into gastric-type protein expression

The origin of gastric metaplasia of the duodenum (GMD) remains enigmatic. We studied expression of mucins and trefoil peptides in GMD to gain insight into its phenotype and origin. We examined duodenal tissue of 95 patients (0 to 83 years old, 26 with gastric Helicobacter pylori infection) for the presence of GMD. Expression was examined immunohistochemically of secretory mucins (MUC2, MUC5AC, MUC5B, and MUC6), trefoil peptides (TFF1, TFF2, and TFF3), and sucrase-isomaltase (SI). GMD, found in 37 patients, correlated positively to gastric H. pylori infection, age, and villus atrophy. MUC2 and TFF3, expressed in normal goblet cells, were absent from 100% and 87% of GMD, respectively. GMD ubiquitously expressed MUC5AC, whereas MUC5AC expression in adjacent goblet cells was closely correlated with the extent of GMD. TFF1, TFF2, and MUC6 were found in 84%, 92%, and 65% of GMD, respectively. MUC5B was absent from epithelium and GMD. SI, expressed by villus enterocytes, was absent from GMD. Brunner's glands ubiquitously expressed MUC5B, MUC6, and TFF2. GMD was characterized by the expression of gastric-type proteins MUC5AC, MUC6, TFF1, and TFF2 and the absence of intestinal markers MUC2, TFF3, and SI. In terms of the location of metaplastic cells, our results suggest that epithelial cells migrating toward villus tips switch to gastric-type secretory cells. Positive correlation with infection suggests an inductive role H. pylori in the development of GMD.     

Isotypes and antigenic profiles of pemphigus foliaceus and pemphigus vulgaris autoantibodies

In this study we systematically characterized isotype profiles and antigenic and tissue specificity of antidesmoglein autoantibodies from patients with pemphigus foliaceus (PF) and pemphigus vulgaris (PV) using enzyme-linked immunoabsorbent assays (ELISA), indirect immunofluorescence (IIF) staining, and immunoblotting (IB). In PF, we found that IgG1 antidesmoglein-1 (Dsg1) reacts with a linear epitope(s) on the ectodomain of Dsg1, while its IgG4 counterpart recognizes a conformational epitope(s). These two subclasses of anti-Dsg1 are both capable of recognizing tissues from monkey esophagus and adult human skin, but IgG1 is not able to react with mouse skin, which may explain why this isotype of anti-Dsg1 failed to induce PF-like lesions in the passive transfer animal model. In mucosal PV patients, we found that both IgG1 and IgG4 only recognized monkey esophagus tissue by IIF, except in one patient, indicating that these antibodies react with a unique conformational epitope(s) that is present in mucosal but not skin tissue. In generalized PV, IgG1 anti-Dsg3 autoantibodies appeared to recognize a linear epitope(s) on the Dsg3 ectodomain. In contrast, IgG4 anti-Dsg3 antibodies recognized both linear and conformational epitopes on the Dsg3 molecule. Interestingly, the IgG1 anti-Dsg3 antibodies failed to react with human and mouse skin tissues, suggesting that this subclass of autoantibodies may not play an essential role in the development of PV suprabasilar lesions. In summary, we conclude that this study further elucidates the pathological mechanisms of PF and PV autoantibodies by revealing their distinct isotype and antigenic profiles. This information may help us to better understand the autoimmune mechanisms underlying the development of pemphigus.     

Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40 degrees C: implications for other inborn errors of metabolism

The peroxisome biogenesis disorders (PBDs), which comprise Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD), represent a spectrum of disease severity, with ZS being the most severe, and IRD the least severe disorder. The PBDs are caused by mutations in one of the at least 12 different PEX genes encoding proteins involved in the biogenesis of peroxisomes. We report the biochemical characteristics and molecular basis of a subset of atypical PBD patients. These patients were characterized by abnormal peroxisomal plasma metabolites, but otherwise normal to very mildly abnormal peroxisomal parameters in cultured skin fibroblasts, including a mosaic catalase immunofluorescence pattern in fibroblasts. Since this latter feature made standard complementation analysis impossible, we developed a novel complementation technique in which fibroblasts were cultured at 40 degrees C, which exacerbates the defect in peroxisome biogenesis. Using this method, we were able to assign eight patients to complementation group 3 (CG3), followed by the identification of a single homozygous c.959C>T (p.S320F) mutation in their PEX12 gene. We also investigated various peroxisomal biochemical parameters in fibroblasts at 30 degrees C, 37 degrees C, and 40 degrees C, and found that all parameters showed a temperature-dependent behavior. The principle of culturing cells at elevated temperatures to exacerbate the defect in peroxisome biogenesis, and thereby preventing certain mutations from being missed, may well have a much wider applicability for a range of different inborn errors of metabolism.     

The prevalence of inherited thrombophilias in a Caucasian Australian population

AIMS: To describe the prevalence of four inherited thrombophilias and their combinations for the first time in a large Caucasian Australian population. METHODS: Newborn screening cards of 883 Caucasian babies born in South Australia in 1986-1999 were de-identified and tested for the following inherited thrombophilic polymorphisms: factor V Leiden (G1691A), prothrombin gene mutation (G20210A), methylenetetrahydrofolate reductase gene (MTHFR) C677T and A1298C, as well as compound heterozygosity for the MTHFR polymorphisms. RESULTS: The birth prevalences of heterozygosity and homozygosity for the four thrombophilic polymorphisms were: factor V Leiden 9.5% and 0.7%, prothrombin gene 4.1% and 0.2%, MTHFR C677T 37.3% and 12.4%, and MTHFR A1298C 38.3% and 11.8%, respectively. Compound heterozygosity for MTHFR C677T and A1298C was seen in 16.6% of the population. Overall, 64.2% and 24.5% of the population studied were homozygous and heterozygous, respectively, for at least one of the four polymorphisms studied. CONCLUSION: Inherited thrombophilic polymorphisms are common in the Caucasian Australian population. Knowledge of the background prevalence of these polymorphisms will allow further study of their associations in future disease research.     

Leaching of oxidation products of aldicarb from greenhouse soils to water courses

When the insecticide/nematicide aldicarb was incubated in three greenhouse soils at 20°C, the transformation rates of the parent compound and its first oxidation product aldicarb-sulfoxide were high, while the amounts of aldoxycarb (=aldicarb-sulfone) remained low. The transformation and movement of aldicarb and its oxidation products in a simplified greenhouse-soil system were simulated with a computation model. When the highest transformation rates were introduced into the computations in combination with high irrigation rates, the computed leaching was only 0.006% of the dose, with a maximum concentration of 0.15g L^–1 in the tile-drain water. With lower transformation rates, the computed leaching was 1.39% of the dose with a maximum concentration of 21.0g L^–1, but in combination with a lower irrigation rate the leaching was computed to be much less. Water from drain pipes and pits was sampled in three greenhouses that had received regular applications of aldicarb: the concentrations measured by high performance liquid chromatography (HPLC) varied from less than 1g L^–1 to more than 30g L^–1. Most of the concentrations measured in water courses in an area with many greenhouses were low, but some were high, ranging up to 21g L^–1. Ways of reducing the discharge of the oxidation products of aldicarb from greenhouse soils to water courses are suggested.