Regulation of apoptosis is impaired in atrophic gastritis associated with gastric cancer.

BACKGROUND: Gastric premalignant conditions, atrophic gastritis (AG) and intestinal metaplasia (IM) are characterized by an increase of proliferation and a reduction of apoptosis in epithelial cells. The epithelial cell kinetics in AG and IM in gastric mucosa adjacent to gastric cancer is still unclear. The aim of this study was to evaluate the epithelial cell turnover and expression of proliferation and apoptosis-related genes in gastric cancer (GC) and adjacent mucosa with atrophic gastritis or intestinal metaplasia (AG/IM GC+), as well as in atrophic gastritis or intestinal metaplasia mucosa of patients without GC (AG/IM GC-) and in control biopsy samples of non-transformed gastric mucosa (Control). METHODS: We selected 58 patients (M: F = 34:24; age range 20-84 years, median 61.06 years) with 4 well defined histological conditions: 20 controls with histological finding of non-transformed gastric mucosa, 20 patients with AG or IM (AG/IM GC-), and 18 patients with intestinal type gastric adenocarcinoma (GC) and AG or IM in the adjacent mucosa (3 cm from the macroscopic tumour margin, AG/IM GC+). We performed an immunohistochemical staining of Ki67 and TUNEL and quantitative RT-PCR to determine the expression of PCNA and Bax/Bcl-2. RESULTS: The immunohistochemical expression of Ki67 and TUNEL in AG/IM GC- was significantly increased compared to not transformed gastric mucosa (p < 0.0001) but not compared to AG/IM in gastric mucosa adjacent to GC. Levels of Bcl-2 were reduced in GC and AG/IM GC- compared to controls as well as in AG/IM GC- compared to AG/IM in mucosa adjacent to GC+ (p < 0.05). Proliferation and apoptosis markers did not correlate with H.pylori status in our study population. CONCLUSIONS: In AG/IM associated with GC, no significant changes in the epithelial cell turnover were detected. Decreased Bcl-2 gene expression signified atrophic gastritis and IM in presence of cancer, as well as intestinal type gastric adenocarcinoma.

Leukotriene receptor expression in esophageal squamous cell cancer and non-transformed esophageal epithelium: a matched case control study.

BACKGROUND: Leukotriene B4 (LTB4R and LTB4R2) and cysteinyl leukotriene receptors (CYSLTR1 and CYSLTR2) contribute to malignant cell transformation. We aimed to investigate the expression of LTB4R, LTB4R2, CYSLTR1 and CYSLTR2 in esophageal squamous cell carcinoma and adjacent non-transformed squamous epithelium of the esophagus, as well as in control biopsy samples from esophageal squamous epithelium of patients with functional dyspepsia. METHODS: Expression of LTB4R, LTB4R2, CYSLTR1 and CYSLTR2 was analyzed by immunohistochemistry (IHC) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in biopsy samples of 19 patients with esophageal squamous cell cancer and 9 sex- and age-matched patients with functional dyspepsia. RESULTS: LTB4R, LTB4R2, CYSLTR1 and CYSLTR2 were expressed in all biopsy samples. Major findings were: 1) protein levels of all leukotriene receptors were significantly increased in esophageal squamous cell cancer compared to control mucosa (p < 0.05); 2) CYSLTR1 and CYSLTR2 gene expression was decreased in cancer tissue compared to control at 0.26-fold and 0.23-fold respectively; 3) an up-regulation of LTB4R (mRNA and protein expression) and a down-regulation of CYSLTR2 (mRNA expression) in non-transformed epithelium of cancer patients compared to control (p < 0.05) was observed. CONCLUSIONS: The expression of leukotriene receptors was deregulated in esophageal squamous cell cancer. Up-regulation of LTB4R and down-regulation of CYSLTR2 gene expression may occur already in normal squamous esophageal epithelium of patients with esophageal cancer suggesting a potential role of these receptors in early steps of esophageal carcinogenesis. Larger studies are warranted to confirm these observations.

[What Must the (Abdominal) Surgeon Know about Experimental Medicine (?) - Translational Research in General (Abdominal) Surgery(Viszeral-)Chirurg & experimentelle Medizin]. / Was muss der (Viszeral-)Chirurg von experimenteller Medizin wissen (?) - translationale Forschung in der (Viszeral-)Chirurgie.

Experimental medicine has evolved tremendously in the last few years. In particular, the introduction of novel techniques, in-vitro models, knock-out/transgenic animals and high-through put analytical methodologies have resulted in a deeper understanding of cellular pathophysiology and diseases. The daily clinical management has benefited by the introduction of biomarkers and targeted therapies. This development has been accompanied by increasing specialisation across all fields of research and medicine. Therefore, clinical-translational research requires a team of competent partners nowadays. The visceral surgeon can contribute significantly to these projects. The present review highlights several aspects of translational research and put chances and potential pitfalls into perspective in context with the work of the visceral surgeon.

[Preventive bone mineral density measurement in postmenopausal women. Differentiation of treatment of recommendations Guidelines of the Umbrella Organization for Osteology ("Dachverband Osteologie")]. / Präventive Knochendichtemessung bei postmenopausalen Frauen. Differenzierung der Therapieempfehlungen der Leitlinien des Dachverbandes Osteologie (DVO).

BACKGROUND: Osteopenia (OP) or osteoporosis (OST) was diagnosed by bone densitometry (DXA) in postmenopausal women free of known skeletal disorders and without acute fracture. DVO guidelines were applied to define therapeutic indication. METHODS: The study included 94 women aged 59-81 years. Fracture or operation ≤12 months, malignant tumor, ovariectomy, and drugs such as cortisone, strontium, fluorides, bisphosphonates, SERMs, estrogens, and steroids were exclusion criteria. The lowest T-score at the spine, femoral neck, or total hip was decisive. The indication for therapy was determined by evaluating age, BMD, and other risk factors. RESULTS: Using the WHO criteria 22.3% (n=21) had normal BMD, 52.1% (n=49) had OP, and 25.6% (n=24) had OST. According to "Dachverband Osteologie" (DVO) guidelines, 28 women (29.8%) of the whole group needed therapy. Of the 28 women receiving therapy, 9 had OP and 19 had OST. Therapy was indicated in 18.4% for OP and 79.2% for OST. CONCLUSION: A preventive measurement of BMD with DXA provides a benefit for postmenopausal women. Combinatory assessment and consideration of other risk factors allows identification of women who might benefit from early treatment.

Helicobacter pylori related and non-related lesions in the stomach.

Gastric lesions may arise in gastric mucosa of patients with gastritis or gastropathies due to different etiopathogenic factors. As most lesions of the stomach result from a chronic infection of gastric mucosa with Helicobacter pylori (H. pylori), a possible classification of gastric lesions based on etiology may distinguish H. pylori-related lesions from those arising in a gastric mucosa not colonized from the bacterium. The repertoire of lesions one may find in the stomach is limited and different pathologies may present with a similar macroscopic aspect. Clinically relevant lesions of the stomach that are or are not associated with H. pylori infection include gastric ulcer, gastric atrophy, gastric neoplasia, and metastasis from other cancers. The detection or exclusion of an H. pylori infection in patients with gastric lesions has important consequences for the clinical management. In the present review we focus on H. pylori-related and non-related peptic lesions in the stomach.

IL-17A and IL-17F gene expression is strongly induced in the mucosa of H. pylori-infected subjects from Kenya and Germany.

Helicobacter pylori infection is the major cause of gastritis. Immunologically, H. pylori gastritis is associated with an infiltration of immune cells into gastric mucosa and the upregulation of various cytokines. Here, we analysed the gene expression of IL-1- and IL-17-related cytokines in regard to H. pylori infection in 85 German and 51 Kenyan patients with reflux-related or dyspeptic symptoms, respectively. Degree of gastritis and density of colonization were assessed histologically in accordance with the updated Sydney classification. Gene expression levels of cytokines IL-1ß, IL-8, IL-18, IL-33, IL-17A, IL-17F and IL-23 as well as IL-23R were analysed by real-time RT-PCR. In both populations, H. pylori-infected individuals had significant higher inflammatory scores for activity and chronicity than H. pylori-negative subjects (P values between 0.006 and <0.0001). IL-8 mRNA was induced up to 6-fold in H. pylori-infected patients (P < 0.05), while the expression levels of IL-1ß, IL-18, IL-23, IL-33 and IL-23R did not differ with respect to the H. pylori status in both groups. Most strikingly, a significant induction of both IL-17A and IL-17F was noted in H. pylori-infected individuals of both ethnic groups. Almost all IL-17F-positive samples revealed co-expression of IL-17A (40/42, 95.2%). Analysing IL-17A and IL-17F transcript levels of these 40 'double-positive' samples, a highly significant positive correlation between both genes was identified (P < 0.001). Taken together, H. pylori infection leads to a strong upregulation of both IL-17A and IL-17F in the gastric mucosa suggesting a regulatory link between both genes.

Host polymorphisms of immune regulatory genes as risk factors for gastric cancer.

The infection of the stomach with the gram-negative bacterium Helicobacter pylori is the main risk factor for the development of gastric cancer (GC). This led to the classification of this germ as "definite carcinogen" by the World Health Organization in 1994. The current model of gastric carcinogenesis is based on the interaction of multiple risk factors including virulence factors of the bacterium (e.g. CagA, VacA), environmental factors (diet, smoking) and host factors (gene polymorphisms). The complex interplay among these factors determines the clinical outcome of the infection leading to at least one of three major diseases in 1 out of 7 infected persons, namely ulcer disease, GC and "mucosa-associated lymphoid tissue" lymphoma in 15 %, 1% and 0.1% of all persons infected with H. pylori, respectively. Recently, an increasing number of genomic polymorphisms, mostly single nucleotide polymorphisms have been identified as risk factors for gastric cancer. Among them are genes encoding for cytokines, pattern recognition receptors, cell cycle-regulators, proteases, HLA-molecules, and enzymes for detoxification. In the last years it has become clear that an uniform "genomic risk pattern" for all GC patients does not exist. Most of these host factors are restricted either to the histological type (intestinal vs. diffuse), ethnical background (particularly Caucasian vs. Asian) and tumor localization (non-cardia vs. cardia cancer). Here, we review the current knowledge about the role of host factors for the gastric carcinogenesis focusing on immune-regulatory genes, in particular on the cytokine interleukin-1beta.

Imatinib for hepatocellular cancer--focus on pharmacokinetic/pharmacodynamic modelling and liver function.

The effects of imatinib are partly mediated by the inhibition of platelet-derived growth factor (PDGF), which is highly expressed in the liver. In this phase-I/II trial pharmacokinetic parameters of imatinib given for hepatocellular cancer were similar to those previously derived from CML patients. The AUC of N-desmethyl-imatinib depended on liver function; the metabolism of imatinib was otherwise comparable to other populations. During short-termed imatinib treatment (4 weeks, 400 mg/d), plasma PDGF significantly decreased. The AUC of N-desmethyl-imatinib could best be attributed to the pharmacodynamic effect of PDGF inhibition (r=-0.679 [95% CI: -0.917 to -0.0868], p=0.031).

Low-dose aspirin reduces the gene expression of gastrokine-1 in the antral mucosa of healthy subjects.

BACKGROUND: Gastrokine 1 (GKN1), one of the most abundant transcripts in normal stomach, is down-regulated by Helicobacter pylori infection. Aspirin (ASA), which is often used for secondary prevention of cardiovascular events, can damage gastric-duodenal mucosa within 1 or 2 h of ingestion. AIM: To study the gastric mucosal expression of GKN1 during acute low-dose ASA consumption. METHODS: Ten H. pylori-negative human volunteers took 100 mg ASA per day for 1 week, and underwent multiple upper GI endoscopies. GKN1 expression was analysed in antral and corpus mucosa by quantitative reverse-transcriptase polymerase chain reaction, western blot and immunohistochemistry (IHC). Gastric mucosal damage was detected endoscopically and histologically. RESULTS: Gastrokine 1 was similarly expressed in both antral and corpus mucosa. The use of low-dose ASA led to a significant decrease (3.07 a.u. vs. 0.23 a.u., P < 0.001) in antrum at day 7, while GKN1 transcript levels in corpus mucosa were slightly elevated (twofold, P < 0.005). Western blot and IHC confirmed these changes at the protein level. Furthermore, IHC revealed a vesicular staining pattern in the cytoplasm for GKN1 that was confirmed by transfected human gastric adenocarcinoma cell line expressing GKN1. CONCLUSION: Our data demonstrated that low-dose ASA downregulates GKN1 expression specifically in antral mucosa.

Low prevalence of Helicobacteraceae in gall-stone disease and gall-bladder carcinoma in the German population.

Colonisation of the hepatobiliary system with bile-resistant Helicobacter spp. has been proposed as a novel risk-factor in the pathogenesis of gall-bladder carcinoma (GBC). There are reports that biliary Helicobacter colonisation is frequent in countries with a high incidence of gall-bladder carcinoma. However, the prevalence of Helicobacteraceae in the gall-bladders of patients with GBC in Germany, a region with a low incidence of GBC, is unknown. Therefore, gall-bladder tissue from 99 patients who had undergone cholecystectomy was tested, including 57 cases of gall-stone disease (GSD), 20 cases of GBC, and 22 control patients. The presence of Helicobacter spp. was investigated by culture, immunohistochemistry and a group-specific PCR targeting the 16S rRNA gene of all currently known Helicobacteraceae. Of the 99 cases investigated, only one patient with GSD was PCR-positive for Helicobacteraceae. For this individual, sequence analysis of the 16S rRNA gene showed that it had homology closest to the 16S rRNA sequence of Helicobacter ganmani. Helicobacteraceae were not detected by culture or immunohistochemistry. The low prevalence of Helicobacteraceae in the gall-bladders investigated suggests that Helicobacteraceae do not play a predominant role in the pathogenesis of GSD and GBC in the German population. The low prevalence could be a possible explanation for a relatively low incidence of GBC in the German population, despite the fact that GSD, the major risk-factor for GBC, is highly prevalent.

Clinical symptom tool that raises the index of suspicion for eosinophilic oesophagitis in adults and drives earlier biopsy for definitive diagnosis.

BACKGROUND: Eosinophilic oesophagitis (EoE) and gastro-oesophageal reflux disease (GERD) present with overlapping symptomatology and it is challenging to distinguish EoE from GERD clinically before endoscopy. AIM: To investigate the prognostic value of a set of clinical symptoms and laboratory values in patients with EoE and GERD. METHODS: In this prospective, single-centre, observational study, we compared clinical and laboratory data from 202 patients with EoE or GERD (10 relevant characteristics). Those characteristics showing potential significance in a univariate analysis were then included in a multivariate analysis. RESULTS: The set of 10 characteristics (10-marker set) was able to distinguish between EoE and GERD with good reliability (correct assignment, i.e. agreement with subsequent EGD, of 94.4%). Reduction of the set to the six statistically and clinically most relevant markers continued to give good reliability (88.9%), and further stepwise reduction led to four-marker sets comprising history of atopy, history of food impaction, proton pump inhibitor refractory symptoms and either immunoglobulin E or peripheral eosinophilia, with correct assignment rates of 91.3% and 85.1% respectively. CONCLUSIONS: We have developed a simple and easily applicable clinical/laboratory marker set that helps to distinguish EoE from GERD earlier in the treatment course, thus guiding the endoscopist to perform biopsies from the oesophagus to ensure the diagnosis. The application of the scoring system is expected to diagnose EoE earlier and avoiding delay of adequate treatment.

The effect of single-dose naproxen on eicosanoid formation in human gastroduodenal mucosa.

BACKGROUND: In animal studies, aspirin and non-aspirin non-steroidal anti-inflammatory drugs contribute to gastroduodenal damage via cyclo-oxygenase inhibition and consecutive leucotriene formation (COX-LOX eicosanoid shunt). AIM AND METHODS: Ten Helicobacter pylori-negative healthy volunteers received a single dose of 500 mg naproxen to address two questions: (i) is there a crosstalk between eicosanoids before medication in the human gastroduodenal mucosa and (ii) can we demonstrate a COX-LOX shunt following single-dose naproxen? RESULTS: Significant correlations in the stomach mucosa before medication were obtained between leucotriene B4 (LTB4) and thromboxane B(2) (TxB(2); r = -0.38, P = 0.05), as well as LTB4 and prostaglandin E(2) (PGE(2); r = 0.71, P < 0.0001). In serum, a >90% inhibition of TxB(2) and PGE(2) occurred within 30 min of naproxen administration. In gastric mucosa, a significant decrease of TxB(2) occurred already at 15 min and preferably in the antrum. For LTB4 there was a non-significant trend towards a transient increase. Mucosal PGE(2) was unchanged in all regions; transcript levels of both cyclo-oxygenases/5-lipoxygenase were unaffected (except for a trend of increasing cyclo-oxygenase-2 in the corpus). CONCLUSIONS: Baseline correlations between LTB4-TxB(2) and LTB4-PGE(2) reflect a crosstalk between these eicosanoids. A COX-LOX shunt; however, cannot be demonstrated following single-dose naproxen in a low-risk population.

Effects of low-dose aspirin on gastric erosions, cyclooxygenase expression and mucosal prostaglandin-E2 do not depend on Helicobacter pylori infection.

BACKGROUND: The mechanisms by which Helicobacter pylori and low-dose aspirin induce gastric damage are not completely elucidated. AIM: To evaluate the effects of low-dose aspirin on gastric damage, mucosal prostaglandin-E(2) levels and cyclooxygenase-enzyme expression in relation to the H. pylori status. METHODS: Twenty healthy volunteers (H. pylori positive, n = 10; H. pylori negative, n = 10) received aspirin 100 mg/die for 1 week. At days 0, 1, 3 and 7, gastric mucosal lesions were studied by oesophagogastroduodenoscopy and histology. COX-1 and COX-2 were determined by immunohistochemistry and reverse-transcriptase polymerase chain reaction, and mucosal prostaglandin-E(2) levels by enzyme-linked immunosorbent assay. Nine H. pylori-positive subjects repeated the protocol after H. pylori eradication. RESULTS: All groups developed a similar number of erosions. COX-1 and COX-2 expression, as well as mucosal prostaglandin-E(2) levels were not influenced by H. pylori status and aspirin medication. Helicobacter pylori-negative and H. pylori-eradicated subjects who developed aspirin-induced erosions had significant lower pre-treatment antral prostaglandin-E(2) levels than those without erosions (3.6 ng/microg vs. 6.3 ng/microg protein and 3.6 ng/microg vs. 6.0 ng/microg protein, respectively, P < 0.01 Mann-Whitney U-test). CONCLUSIONS: In healthy subjects, low-dose aspirin for 1 week does neither affect cyclooxygenase expression nor mucosal prostaglandin-E(2) levels. Antral prostaglandin-E(2)-basal levels appear to be critical for development of aspirin-induced gastric damage in subjects without H. pylori infection.

Impact of biomarkers on disease survival and progression in patients treated with octreotide for advanced hepatocellular carcinoma.

BACKGROUND: Current determination of prognosis for advanced hepatocellular carcinoma (HCC) is mainly based on clinical assessment. We aimed to determine the impact of biomarkers as predictive factors for HCC progression and survival during octreotide-based treatments. PATIENTS AND METHODS: We included patients who had been prospectively randomised to receive either octreotide (30 mg) alone monthly (n = 39) or in combination with rofecoxib (up to 50 mg bid daily, n = 32) for a minimum of 6 months, or until death occurred. RESULTS: Overall median survival (154 days) and median time to progression (94 days) were not different for both treatments and the biomarkers investigated (VEGF-A, IGF-1, PGE-2, ET-A) were similarly distributed amongst treatment groups. Combined univariate group analysis revealed that survival was decreased for an uptake ratio of > 2 on initial octreoscan (P = 0.05); baseline serum VEGF-A and IGF-1 were further significantly associated with survival. On multivariate analysis, uncorrected serum VEGF-A appeared to be the most significant predictor for tumor progression and survival. CONCLUSIONS: Biomarkers, in addition to established tumor markers, are independent predictors of tumor progression and survival in patients with advanced HCC treated with octreotide. Furthermore, the involvement of VEGF-A implies the inhibition of angiogenesis as a potential mechanism of action for this drug.

Helicobacter pylori infection is associated with a reduced risk of developing eosinophilic oesophagitis.

BACKGROUND: Eosinophilic oesophagitis (EoE) represents a chronic immune-antigen-mediated allergic disease of the oesophagus of still unknown aetiology. Environmental exposure has been postulated to play a pathogenetic role. Helicobacter pylori (H. pylori) infection has been inversely associated with allergic diseases including atopic dermatitis, asthma and allergic rhinitis and H. pylori may play a protective role in these conditions. Little is known about the relationship between EoE and H. pylori. AIM: To investigate in a case-control study whether H. pylori infection is associated with a reduced risk of developing EoE. METHODS: H. pylori infection was evaluated by serology in 58 [11(19%) female, 47 (81%) male, median age: 36.5 years, range 20-72 years] patients with a clinical and histologically proven diagnosis of EoE and 116 age and sex-matched controls (1 case: 2 controls). Antibodies against H. pylori were identified by enzyme-linked immunosorbent assay. Patients with H. pylori-specific IgG ≥ 30 enzyme immunounits were classified as H. pylori-positive. RESULTS: 3/58 (5.2%) patients with EoE had serological evidence of H. pylori infection (EoE - H. pylori current infection) and 5/58 (8.6%) reported prior eradication therapy for H. pylori infection (EoE - H. pylori former infection). The control group demonstrated significantly higher seroprevalence of H. pylori (37.9%, P < 0.0001) when compared to patients with EoE. EoE was inversely associated with H. pylori infection [odds ratio (OR) 0.24, 95% confidence interval (CI) 0.11-0.50]. CONCLUSION: Helicobacter pylori infection is inversely associated with EoE. Our results may contribute to further understanding the pathogenesis and evolving aetiology of EoE.

Impact of the angulus biopsy for the detection of gastric preneoplastic conditions and gastric cancer risk assessment.

BACKGROUND: Gastric atrophy and intestinal metaplasia (IM) are preneoplastic conditions in the development of gastric cancer. Histopathological assessment is based on the updated Sydney system and superordinate staging systems, operative link on gastritis assessment (OLGA) and operative link on gastritis assessment using IM (OLGIM), all requiring a biopsy from the incisura angularis (angulus). AIM: To determine the value of the angulus biopsy for the detection of preneoplastic conditions and cancer risk evaluation using OLGA and OLGIM prospectively. METHODS: Biopsies from antrum (2), angulus (1) and corpus (2) were obtained from 213 patients (age 19-94 years, median 54 years, female to male ratio 138:75) undergoing upper endoscopy. Histological assessment according to the updated Sydney system, OLGA and OLGIM staging was performed by gastrointestinal pathologists. Statistical analysis used exact confidence limits for dichotomous variables and repeated measurement analysis of variance. RESULTS: 8% of the cases with atrophic gastritis and 3% with IM (17 vs 6/213) would have been missed without the angulus biopsy. More patients were diagnosed with a preneoplastic condition when the angulus biopsy was considered (13.1%, CI 8.9% to 18.4%), but the grade of atrophy, if present at both sides, did not vary significantly in angulus and antrum. OLGA and OLGIM scores dropped significantly when recalculated without the angulus (difference in means±SD 0.131±0.402 and 0.075±0.313, respectively). The impact on the identification of high-risk stages is limited. CONCLUSIONS: The angulus biopsy adds to the detection of mild gastric atrophy in particular. It allows identifying a small additional number of patients with high-risk gastritis.

The main neutral aminopeptidase activity of human lymphoid tumour cell lines does not originate from the aminopeptidase N-(APN; CD13) gene.

Lymphocytes and related cell lines are predominantly CD13-negative, however, there are reports describing neutral aminopeptidase activity in or on these cells. The aim of this study was to answer the question, whether this activity originates from APN-gene expression. The total cellular activities (Ala-pNA hydrolysis) of lymphoid cell lines are up to 15 times higher than that of normal lymphocytes. Despite weak or lacking CD13 surface expression all lymphoid cell lines tested contain APNmRNA as quantified by competitive RT-PCR as well as low enzymatic activity in their particulate fractions. By isoelectric focusing two enzyme species with isoelectric points of 5.4 or between 3.5 to 4.8, respectively, were detected. To investigate whether these activities result from APN-gene we established transfectants lacking cellular APN expression of the CD13-positive histiocytic cell line U937 and the CD13-negative T-cell line H9. Studies on these transfectants proved (I) that the main neutral aminopeptidase activity expressed in lymphoid cells is definitively not related to APN and (II) that APN is also expressed in lymphoid cells, although on a low level only.

Quantification of aminopeptidase N mRNA in T cells by competitive PCR.

The aminopeptidase N (CD13, EC 3.4.11.2) is a well-characterized surface molecule expressed in a variety of cell types and species. Recent data indicate an expression of the APN mRNA and the corresponding aminopeptidase activity in human peripheral T cells and related cell lines as well. Here, the sensitive method of competitive PCR was used to quantify low amounts of APN mRNA in T cell lines. An APN cDNA fragment enshortened by a deletion of 87 bp was used as an internal APN-specific standard. The myelo-monocytic cell line U937 and the lymphoid T cell lines HuT78 and H9 contain 2.3 x 10(7), 5.9 x 10(6) and 5.6 x 10(6) copies/micrograms total RNA, corresponding to 160, 70 and 50 copies/cell, respectively. These data have been confirmed by determination of the APN activity, that represents a fraction only of the total cellular neutral aminopeptidase activity in hematopoetic cells. In the case of the CD13-positive cell line U937, approximately 60-70% of the total neutral aminopeptidase activity could be attributed to APN. In contrast, only a minor fraction (5-20%) of the cellular neutral aminopeptidase activity in the T cell lines H9 and HuT78 represents APN. The results suggest that APN gene expression within the hematopoetic system is not restricted to myelo-monocytic cells, instead a low APN expression may be a common feature of lymphocytes, at least of T cells, too.

The activation-dependent induction of APN-(CD13) in T-cells is controlled at different levels of gene expression.

Recently, it was shown that aminopeptidase N (E.C. 3.4.11.2, CD13) is up-regulated during mitogenic stimulation of peripheral T-cells. In this study, we demonstrate that the half-life of APN mRNA was considerably prolonged in these cells leading to a 2.7-fold increase of APN transcript level. The apparent half-life time of the APN transcript was investigated by the RNA synthesis inhibitor-chase method using actinomycin D. The steady-state APN mRNA levels was determined by a competitive RT-PCR. The half-lives estimated in resting T-cells, natural killer cells and permanently growing tumour cells varied between 3.5 and 6 h. Finally, nuclear run-on assays revealed that the APN gene expression of stimulated T-cells is controlled by increased promoter activity as well. These studies suggest a control of APN gene expression at the post-transcriptional level in addition to promoter-mediated regulation.

Rapid mitogen-induced aminopeptidase N surface expression in human T cells is dominated by mechanisms independent of de novo protein biosynthesis.

The membrane bound metalloprotease aminopeptidase N (APN, CD13, EC 3.4.11.2) is a well established marker of normal and malignant cells of the myelo-monocytic lineage. It is also expressed by leukaemic blasts of a small group of patients suffering from acute or chronic lymphoid leukaemia. Recently, the expression of the APN gene in T cell lines as well as the induction of APN gene and surface expression in human peripheral T cells by mitogenic activation have been demonstrated. Here, by means of cytofluorimetric analysis evidence is provided, that the induction of APN surface expression is partially resistent to the action of the inhibitors of protein biosynthesis, puromycin and cycloheximide, and is not prevented by tunicamycin, an inhibitor of glycosylation. These data suggest that the rapid mitogen-induced surface expression of APN, detectable 20 hours after stimulation is dominated by mechanisms not dependent on de novo protein biosynthesis or glycosylation. As shown by simultaneous analyses, the inhibitors used did also differently modify the induction of surface expression of other inducible glycosylated leukocyte surface antigens, namely CD25, CD69 and CD95.