[Clinical utility of EUS-FNA in upper gastrointestinal and mediastinal disease]. / Klinische Wertigkeit der endosonographischen Feinnadelpunktion bei Erkrankungen des oberen Gastrointestinaltrakts und Mediastinums.

BACKGROUND: Endoscopic ultrasound (EUS)-guided fine-needle aspiration biopsy (EUS-FNA) is increasingly used for the diagnosis of malignant and benign disease in the region of the upper GI tract. We prospectively investigated the clinical accuracy and safety of this method in unselected patients under routine conditions. PATIENTS AND METHODS: 101 consecutive patients (median 61.5 years; 56 female) were enrolled in the study, in whom a total of 106 tissue biopsies were obtained by using EUS-FNA. Major indications for EUS-FNA were suspicious lesions located in the mediastinum, esophagus, stomach, pancreas, liver, biliary system, adrenals or retroperitoneum. A longitudinal echoendoscope (HITACHI FG-34UX) equipped with a standard 22G -aspiration needle was used. The aspirated specimens were analyzed further by using standard cytology and/or histology. Lymph-node biopsies were additionally subjected to flow-cytometry (FACS-light-chain restriction). Surgery was used for reference (where available). In the remaining cases the final diagnosis obtained by the clinical course and all available imaging and histologic informations (ultrasound, CT, MRT) was used for reference. RESULTS: EUS-FNA caused no serious complications. In 6/106 specimen (5.6 %) no sufficient cell material could be aspirated. In the remaining 100 specimens EUS-FNA reached an overall sensitivity of 78 % and a specificity of 100 %, while the accuracy was 89 % and the positive and negative predictive values were 100 % and 81 %, respectively. The greatest diagnostic accuracy was achieved in mediastinal and retroperitoneal lesions, while the accuracy of EUS-FNA in pancreatic lesions and perigastric lymph nodes was distinctly smaller (<80 %). Addition of FACS studies in patients with suspected malignant lymphoma increased the diagnostic accuracy in the small number of patients included in the study. CONCLUSION: EUS-FNA improves the tissue-based diagnosis of suspicious lesions in locations that are difficult to access (e. g., posterior mediastinum). EUS-FNA is safe, while its diagnostic accuracy is relatively high. Our preliminary data suggest that flow-cytometry may improve the fine-needle based diagnosis of non-Hodgkin s lymphoma, which should be further investigated.

[Diagnostic utility of capsule endoscopy in occult gastrointestinal bleeding]. / Diagnostik okkulter Blutungen des oberen Gastrointestinaltraktes durch die Videokapselendoskopie.

BACKGROUND AND OBJECTIVE: The video-capsule endoscopy (CE) of the small intestine is a novel innovative procedure for outpatient use that can detect even small lesions of the mucosa of the small intestine. Aim of this retrospective clinical study was to evaluate the diagnostic value of CE in a clinical routine setting. PATIENTS AND METHODS: Between July 2001 and October 2002 we investigated 42 patients with suspected gastrointestinal bleeding by CE. In all patients, the previous upper and lower endoscopy work-up was normal. In some cases additional procedures such as bloodpool scintigraphy, angiography, small-bowel enteroclysis or push-enteroscopy were performed. RESULTS: CE detected relevant pathological findings in 23 out of 42 Patients (55 %). The majority of findings in the CE consisted of angiodysplasia (n = 16), ulcer and haemorrhagical erosions (n = 10), one Ulcus Dieulafoy and additional polyps of the small intestine (n = 2). In 4 cases an inflammatory small-bowel disease was detected. These findings could be confirmed by Re-endoscopy. The information provided was helpful to direct further diagnostic and treatment options. In 14 cases (33 %) CE-findings steered additional diagnostic and therapeutic steps. We conclude that CE is safe and has a high diagnostic yield. CONCLUSION: M2A video CE is likely to become an integral part of the algorithm of diagnostic of occult gastrointestinal bleeding after exclusion of other causes of anemia and negative upper and lower endoscopy work-up.

Endoscopic ultrasound-guided fine-needle aspiration biopsy of liver lesions: histological and cytological assessment.

BACKGROUND AND STUDY AIMS: EUS-guided fine-needle aspiration biopsy (EUS-FNA) is used increasingly for the diagnosis of mediastinal, biliopancreatic, and gastric tumors. However, little is known about EUS-FNA in hepatic lesions and the best method for tissue analysis. We assessed EUS-FNA combined with histological and cytological evaluation in selected patients. PATIENTS AND METHODS: 41 patients (66 +/- 7 years) were prospectively studied, 33 of whom had clinical findings suggestive of liver malignancies. Selection for EUS-FNA was based on an increased risk of bleeding from percutaneous biopsy (coagulopathy, cirrhosis, ascites, aspirin intake; n = 15), presence of small liver tumors < 2 cm (n = 12), or liver lesions found incidentally (n = 14). Transgastric EUS-FNA of lesions located in accessible liver segments was performed using the Hitachi FG-34UX longitudinal echo endoscope and a 22-G aspiration needle. Specimens were submitted separately for standard cytological and histological evaluation. In the case of malignancies, findings at surgery with histological examination, endoscopy, or computed tomography (CT)-guided biopsy of the primary cancer served as reference results (n = 33), while in benign disorders, a combination of imaging studies (Magnetic Resonance Tomography , scintigraphy) and the clinical follow-up, as summarized in the physician's report, was used as reference. RESULTS: EUS-FNA provided appropriate biopsy specimens in 40/41 patients. It was not possible to aspirate sufficient material in one patient. On average, 1.4 needle passes were necessary to obtain sufficient amounts of tissue. With regard to malignancy, the combination of histological and cytological examination had a sensitivity of 94%, specificity of 100%, negative predictive value (NPV) of 78%, and positive predictive value (PPV) of 100%. Tissue diagnoses were in agreement in 27/41 patients (65%). In the remaining patients, only the cytological examination identified six lesions correctly, while the histological assessment was correct in another seven patients. Malignant lesions were correctly identified by cytology in 24/33 (73%) patients, while histology alone was diagnostic for malignancy in 27/33 (82%) patients. When both modalities were combined, 31 out of 33-malignancies (94%) were correctly diagnosed. Minor complications occurred in two patients and consisted of self-limiting local bleeding. CONCLUSIONS: EUS-FNA of liver tumors is a powerful, reliable, and safe procedure for the diagnosis of malignant liver lesions. Optimal diagnostic results are achieved by combining cytological with histological assessment. Hence, EUS-FNA is an alternative to percutaneous biopsy, particularly in patients at risk of bleeding or with small lesions of the liver.

Endoscopically assisted video capsule endoscopy of the small bowel in patients with functional gastric outlet obstruction.

Since the introduction in 2001 of M2A video capsule imaging of the small bowel in humans, this technique has been used increasingly in patients with disorders of the small bowel. In particular the assessment of small obscure gastrointestinal bleeding sources and the detection of shallow inflammatory lesions in the small bowel, have been greatly facilitated by this novel imaging procedure. We report two cases of patients with obscure gastrointestinal bleeding, in whom normal passage of the capsule through the antroduodenal junction was inhibited. This was because of delayed gastric emptying in both patients, which was presumably caused by functional impairment of pyloric motility. To facilitate capsule transport into the small bowel, after swallowing the capsule each patient underwent unsedated upper gastrointestinal endoscopy during which the capsule was grasped with a polypectomy snare, directly transported through the pylorus, and finally released upon arrival in the second portion of the duodenum. Capsule recordings revealed the source of bleeding in both patients and their medical or surgical treatment was subsequently escalated. Capsule imaging of the small bowel facilitated by esophagogastroduodenoscopy (EGD) is safe, and can be applied when patients have functional disorders of pyloric motility.

M.(phi)BssHII, a novel cytosine-C5-DNA-methyltransferase with target-recognizing domains at separated locations of the enzyme.

In all cytosine-C5-DNA-methyltransferases (MTases) from prokaryotes and eukaryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order. In addition, one variable region, which includes the target-recognizing domain(s) (TRDs) characteristic for each enzyme, has been localized in one region between the same blocks of these conserved elements. This conservation in the order of conserved and variable sequences suggests stringent structural constraints in the primary structure to obtain the correct folding of the enzymes. Here we report the characterization of a new type of a multispecific MTase, M.(phiphi)BssHII, which is expressed as two isoforms. Isoform I is an entirely novel type of MTase which has, in addition to the TRDs at the conventional location, one TRD located at a non-canonical position at its N-terminus. Isoform II is represented by the same MTase, but without the N-terminal TRD. The N-terminal TRD provides HaeII methylation specificity to isoform I. The TRD is fully functional when engineered into either the conventional variable region of M.(phiphi)BssHII or the related monospecific M.phi3TII MTase. The implications of this structural plasticity with respect to the evolution of MTases are discussed.

Electron microscopy and capillaroscopically guided nailfold biopsy in connective tissue diseases: detection of ultrastructural changes of the microcirculatory vessels.

The aims of the study were to describe and compare the frequency and nature of histologically detectable microvascular lesions in patients with various connective tissue diseases (CTD). An electron microscopic examination of specimens obtained by the technique of capillaroscopically guided nailfold biopsy was performed in 52 patients with CTD [nine systemic lupus erythematosus (SLE), eight mixed CTD, 18 scleroderma, 17 undifferentiated CTD] and 27 controls. The microvascular changes most frequently observed by electron microscopy were multilayering of the basal lamina (approximately 70% of the CTD patients), an increased amount of perivascular connective tissue, perivascular oedema formation, and an increased number of perivascular fibroblasts and mast cells (each in 30-37% of the CTD patients). In contrast, no particular histopathological feature was found in > 25% of the controls, multilayering (22.6%) being the most frequently observed. Comparing the different conditions studied, there were distinct differences in the frequency and nature of the histologically observed microvascular changes. In particular, SLE seems to be based on a separable type of vasculopathy consisting of significantly less frequent microvascular abnormalities. In conclusion, ultrastructural abnormalities of the microvascular system are a frequent finding in CTD. Electron microscopic examination of specimens obtained by capillaroscopically guided nailfold biopsy is able to disclose histopathological differences between defined entities. Therefore, this approach may be a useful tool to gain further insights into potentially separable aetiopathological mechanisms of the various types of CTD.

M.BssHII, a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties.

A new multispecific cytosine-C5-DNA-methyltransferase (C5-MTase), M.BssHII, was identified in Bacillus stearothermophilus H3. The M.BssHII gene was cloned and sequenced. The amino acid sequence deduced shows the characteristic building plan of a C5-MTase. By sequencing bisulfite-treated DNA methylated by M.BssHII and by restriction enzyme analysis, we defined the following methylation targets of M.BssHII: ACGCGT/CCGCGG (MluI/SacII), PuGCGCPy (HaeII), PuCCGGPy (Cfr10I) and GCGCGC (BssHII). The relative location of the specificity determinants in the C5-MTase was derived from the analysis of M.BssHII derivatives carrying deletions within the variable region "V" and chimeric C5-Mtases constructed between M.BssHII and the related monospecific enzyme M.phi3TII. Four of the M.BssHII specificities (MluI, SacII, Cfr10I and BssHII) could be associated with amino acid segments within the variable region "V". The determinant for HaeII activity had to be assigned to sequences defining the enzyme core, the first example of a C5-MTase in which a sequence-specific methylation potential is mediated by structures outside of the variable region. Another intriguing result came from the analysis of one particular chimera made between M.BssHII and M.phi3TII. This construct showed a relaxation of the methylation capacity, both with respect to the target recognized and the targeting of methylation within this sequence.

Exact size and organization of DNA target-recognizing domains of multispecific DNA-(cytosine-C5)-methyltransferases.

A large portion of the sequences of type II DNA-(cytosine-C5)-methyltransferases (C5-MTases) represent highly conserved blocks of amino acids. General steps in the methylation reaction performed by C5-MTases have been found to be mediated by some of these domains. C5-MTases carry, in addition at the same relative location, a region variable in size and amino acid composition, part of which is associated with the capacity of each C5-MTase to recognize its characteristic target. Individual target-recognizing domains (TRDs) for the targets CCGG (M), CC(A/T)GG (E), GGCC (H), GCNGC (F) and G(G/A/T)GC(C/A/T)C (B) could be identified in the C-terminal part of the variable region of multispecific C5-MTases. With experiments reported here, we have established the organization of the variable regions of the multispecific MTases M.SPRI, M.phi3TI, M.H2I and M.rho 11SI at the resolution of individual amino acids. These regions comprise 204, 175, 268 and 268 amino acids, respectively. All variable regions are bipartite. They contain at their N-terminal side a very similar sequence of 71 amino acids. The integrity of this sequence must be assured to provide enzyme activity. Bracketed by 6-10 'linker' amino acids, they have, depending on the enzyme studied, towards their C-terminal end ensembles of individual TRDs of 38 (M), 39 (E), 40 (H), 44 (F) and 54 (B) amino acids. TRDs of different enzymes with equal specificity have the same size. TRDs do not overlap but are either separated by linker amino acids or abut each other.

M.BssHII: a new multispecific C5-DNA-methyltransferase.

M.BssHII is a new multispecific C5-DNA-methyltransferase recognizing five different targets. As the enzyme has been isolated from a thermophilic Bacillus, the protein should show enhanced intrinsic thermostability and therefore be a promising candidate for crystallizing a multispecific MTase.

BsuCI, a type-I restriction-modification system in Bacillus subtilis.

A type-I R-M system was identified in B. subtilis. The genes comprising the system have striking similarity to type-I R-M systems observed in Enterobacteriaceae.

Deletion of chromosome arm 17p DNA sequences in pediatric high-grade and juvenile pilocytic astrocytomas.

In adults, loss of heterozygosity for DNA on 17p has been shown in high-grade anaplastic astrocytomas (AAs) and glioblastomas multiforme (GMs), and mutation of the TP53 tumor suppressor gene has been reported in all grades of astrocytomas. Little is known, however, about 17p deletion and TP53 mutation in juvenile pilocytic astrocytomas (JPAs), the most common low-grade tumors seen in children. To elucidate the genetic characteristics of pediatric high-grade astrocytomas and JPAs, we performed restriction fragment length polymorphism analysis with probes derived from 17p and TP53 mutational studies in 28 tumor specimens. Telomeric chromosome arm 17p markers 144-D6 and ABR were lost in 6 (75%) of 8 informative tumors classified as high-grade (7 AAs, 1 GM) and in 2 (10%) of 20 informative JPAs. Loss of 17p probes centromeric to the TP53 gene were also detected in 3 AAs and 5 JPAs. Four of the 6 (66%) JPAs with losses of 17p DNA sequences recurred rapidly despite aggressive therapy, whereas only 5 of the other 14 (36%) recurred. Mutation of the TP53 gene was detected by polymerase chain reaction and denaturing gradient gel electrophoresis in only 1 JPA and 1 AA. These tumors were also examined for MDM2 gene amplification as an alternate inactivation mechanism for TP53 gene function: no instances of alteration were identified. These results suggest that a gene or genes in addition to TP53 on 17p may be involved in the etiology or progression of high-grade astrocytomas and aggressive JPAs in children.(ABSTRACT TRUNCATED AT 250 WORDS)

Physical mapping of chromosome 17p13.3 in the region of a putative tumor suppressor gene important in medulloblastoma.

Deletion mapping of a medulloblastoma tumor panel revealed loss of distal chromosome 17p13.3 sequences in tumors from 14 of 32 patients (44%). Of the 14 tumors showing loss of heterozygosity by restriction fragment length polymorphism analysis, 14 of 14 (100%) displayed loss of the telomeric marker p144-D6 (D17S34), while a probe for the ABR gene on 17p13.3 was lost in 7 of 8 (88%) informative cases. Using pulsed-field gel electrophoresis, we localized the polymorphic marker (VNTR-A) of the ABR gene locus to within 220 kb of the p144-D6 locus. A cosmid contig constructed in this region was used to demonstrate by fluorescence in situ hybridization that the ABR gene is oriented transcriptionally 5' to 3' toward the telomere. This report provides new physical mapping data for the ABR gene, which has not been previously shown to be deleted in medulloblastoma. These results provide further evidence for the existence of a second tumor suppressor gene distinct from p53 on distal chromosome 17p.