A biopsy-based quick test in the diagnosis of duodenal hypolactasia in upper gastrointestinal endoscopy.

BACKGROUND AND STUDY AIMS: The usefulness of a new quick test for endoscopic diagnosis of adult-type hypolactasia was tested in duodenal biopsies. In this test, an endoscopic biopsy from the postbulbar duodenum is incubated with lactose on a test plate, and a color reaction develops within 20 min as a result of hydrolyzed lactose (a positive result) in patients with normolactasia, whereas no reaction (a negative result) develops in patients with severe hypolactasia. PATIENTS AND METHODS: Two postbulbar duodenal biopsies were taken from 80 prospectively enrolled adult outpatients with dyspepsia. The biopsies were used for the Quick Lactase Test (Biohit PLC, Helsinki, Finland) and in biochemical disaccharidase (lactase, sucrase, and maltase) assays. In addition, the C/T (-13,910) genotype was determined from DNA extracted from gastric antral biopsies using polymerase chain reaction sequencing in genomic analysis of adult-type hypolactasia. RESULTS: Twenty-one of 22 patients (95 %; 95 % CI, 87 - 100 %) with biochemical lactase activity < 10 U/g protein, but none of the 58 patients with lactase activity of 10 U/g protein or more had a negative result in the Quick Lactase Test. Seven of the 80 patients (9 %; 95 % CI, 3 - 15 %) had a Quick Lactase Test result that indicated mild hypolactasia (a mild color reaction). All patients with celiac disease (n = 6) had a negative Quick Lactase Test result. Nine of 74 patients (six patients with celiac disease were excluded) had a CC (-13,910) genotype in genomic testing, indicating adult-type hypolactasia. All of them had negative test results with the Quick Lactase Test. Twenty-six patients had a TT genotype, indicating normolactasia, and none of these patients had a negative test result in the Quick Lactase Test. Six of 39 patients (15 %; 95 % CI, 4 - 27 %) with a CT genotype had a negative result in the Quick Lactase Test. CONCLUSIONS: The Quick Lactase Test effectively identifies patients with severe duodenal hypolactasia. In comparison with CC (adult-type hypolactasia) and TT individuals (normolactasia), the sensitivity and specificity of the Quick Lactase Test result was 100 %. In comparison with biochemical lactase assays, the sensitivity and specificity of a negative Quick Lactase Test for indicating hypolactasia (lactase activity < 10 U/g protein) were 95 % (95 % CI, 87 - 100 %) and 100 %, respectively.

Serum levels of amidated gastrin-17 and pepsinogen I in atrophic gastritis: an observational case-control study.

BACKGROUND: Helicobacter pylori infection is often diagnosed with non-endoscopic methods, such as serology or breath or antigen stool tests. These tests provide information on the presence or absence of the H. pylori gastritis only. We investigated whether atrophic gastritis can be diagnosed and typed non-endoscopically if the serum levels of pepsinogen I (S-PGI) and gastrin-17 (S-G-17) are assayed in connection with H. pylori testing. METHODS: The present investigation is an observational case-control study comprising 100 selected dyspeptic outpatients with (cases) or without (controls) advanced (moderate or severe) atrophic gastritis. Before the blood tests, all patients underwent a diagnostic gastroscopy with multiple biopsies. The series of cases includes 56 patients. Eight had an advanced antrum limited atrophic gastritis, 13 had resected antrum (in two of whom the corpus mucosa in the stump was atrophic), and 30 had corpus-limited atrophic gastritis. Four patients had an advanced atrophic gastritis in both the antrum and corpus (multifocal atrophic gastritis), and the whole stomach was removed in one patient. Twenty of the 44 controls had a non-atrophic H. pylori gastritis. Both the antrum and corpus were normal and healthy in 24 patients. The S-PGI and S-G-17 were determined with EIA methods using monoclonal antibodies to PGI and amidated G-17. Postprandial S-G-17 (S-G-17prand) was measured 20 min after a protein-rich drink. The H. pylori antibodies were assayed with a polyclonal EIA method. RESULTS: A low S-PGI (<25 microg/l; an empirical cut-off with best discrimination) was found in 31 of 37 patients (84%) with and in 3 of 63 patients (5%) without corpus atrophy in the biopsy specimens. A low S-G-17prand (<5 pmol/l) was found in all 8 patients with H. pylori-associated antral atrophy and in 11 of 14 patients (79%) with resected antrum but in 3 of 20 control patients (15%) with H. pylori-related non-atrophic gastritis. Median and mean values of both S-G-17prand and S-PGI decreased with increasing grade of antral and corpus atrophy, respectively. Among all patients with atrophic gastritis (multifocal atrophic gastritis, or atrophic gastritis limited to antrum or corpus) or resected stomach, 50 of 56 patients (89%; Cl 95%: 81%-97%) had a low S-PGI and/or a low S-G-17prand with positive H. pylori serology. Such low values werc found in 3 of the 44 control patients (7%; CI 95%: 0%-14%). CONCLUSIONS: Low serum levels of G-17prand and PGI are conceivable biomarkers of atrophic antral and corpus gastritis, respectively. A low S-G-17prand is a sign of the multifocal or antrum-limited atrophic gastritis in patients infected with H. pylori.

Specialists as consultants to GPs. Private sector services as an alternative way of organising consultant services in health care.

OBJECTIVE: To assess the effect on referral rate when GPs are given a better opportunity to send their patients to specialists for consultation. DESIGN: Intervention study with a control group. SETTING AND SUBJECTS: A 34-month experiment in the City of Turku. The experimental group consisted of 10 GPs working in municipal health centres with a list system and serving 23000 residents. As a control group, there were four GPs without a list system serving 10800 residents. OUTCOME MEASURES: GP visits and their referrals to specialists. RESULTS: The number of patient visits was higher among GPs in the experimental group than among those in the control group. During the experiment, the referral rate of GPs in the experimental group increased from 5.7% to 6.8%, but there was no change among the control GPs. Of all referrals to specialists, the share to the private sector increased from 5% to 35%, while at the same time the share to the hospital outpatient clinic decreased. CONCLUSIONS: An enhanced possibility to consult private specialists increases the number of referrals, but there are no consequent changes in the relative shares of the consulted specialties.

Tenascin and fibronectin expression in human mesothelial cells and pleural mesothelioma cell-line cells.

Fibronectin (Fn) and tenascin (Tn) are two major extracellular matrix (ECM) glycoproteins that may have important roles both in fibrotic lung diseases and in lung tumors. The significance of Fn and Tn in human pleural mesothelial cells and pleural diseases is unclear. Transformed human pleural mesothelial cells (Met5A), primary cultures of mesothelial cells, and cultured mesothelioma cell lines were investigated for Fn and Tn immunoreactivity. Mesothelial cells were exposed for 48 to 96 h to transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), amosite asbestos fibers, or oxidants (H2O2 and menadione, a compound that auto-oxidizes to produce superoxide). Immunofluorescence and Western blotting with monoclonal anti-Fn and anti-Tn antibodies, and Northern blotting with a complementary DNA (cDNA) probe for Tn showed that mesothelial cells are capable of producing Fn and Tn. The mRNA level and immunoreactivity of Tn was enhanced by TGF-beta and TNF-alpha, whereas Fn was intensified only by TGF-beta. A wide range of amosite, H2O2, or menadione concentrations had no clear effect on Fn or Tn reactivity. Fn and Tn were present at low or undetectable concentrations in five of six mesothelioma cell lines, whereas the organization of Fn immunoreactivity in these cell lines was variable. Furthermore, results obtained with the tumor tissue of these same mesothelioma patients suggested that Fn and Tn expressions do not necessarily parallel either each other or results obtained with the cultured cells.

Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5, and migrate on the newly deposited basement membrane.

We studied the adhesion mechanism of pancreatic carcinoma using in vitro adhesion and migration assays of stable cell lines and tumors grown from these cell lines in nude mice. We also compared the results with the expression profiles of laminins and their receptors in pancreatic carcinomas to evaluate the relevance of these mechanisms in vivo. All of the cell lines preferably adhered to laminin-5, irrespective of their capability to synthesize laminin-5. Cell migration was studied in the presence of hepatocyte growth factor, as it increased the speed of migration manyfold. Herbimycin A treatment and antibodies against the beta 1 and alpha 3 integrin subunits and laminin alpha 3 chain almost entirely blocked cell migration of the BxPC-3 cell line, whereas migration was nearly unaffected by RGD peptide and only moderately inhibited by antibody against the alpha 6 integrin subunit. Indirect immunofluorescence microscopy of wounded BxPC-3 cells suggested a rapid endocytosis of alpha 3 integrin subunit in the cells at the margin of the wound and a rapid, polarized rearrangement of the alpha 6 beta 4 integrin. Especially HGF-treated cultures showed a prominent cytoplasmic reaction for laminin-5 at the margin of the wound. Xenografted cells formed tumors that produced and deposited the same laminin chains as the in vitro cultures. Frozen sections of human pancreatic carcinomas showed reactivity for laminin chains suggestive for expression of laminin-1 and laminin-5. Both xenografted tumors and human pancreatic carcinomas also showed stromal reactivity for laminin-5. Electron microscopy of the human tumors suggested that this was due to an abundant reduplication the basement-membrane-like material around the nests of malignant cells. Our results suggest that pancreatic carcinomas synthesize and deposit laminin-5 in the basement membrane in an abnormal manner. Invading cells adhere to this newly produced basement membrane and migrate on it by using the alpha 3 beta 1 integrin receptor recognizing laminin-5.

Neuronal differentiation in SH-SY5Y human neuroblastoma cells induces synthesis and secretion of tenascin and upregulation of alpha(v) integrin receptors.

Human SH-SY5Y neuroblastoma cells were induced to neuronal differentiation by using 12-0-tetradecanoylphorbol-13-acetate (TPA) and retinoic acid (RA). Both treatments rapidly induced long neurites and increased the content of neurofilaments as shown by immunocytochemistry and immunoblotting. Immunoprecipitation and immunoblotting of the culture medium with monoclonal antibodies demonstrated a rapid onset of synthesis and secretion of Mr 280,000 tenascin (Tn) polypeptide with TPA and both Mr 280,000 and 190,000 Tn polypeptides with RA and an increased secretion of extradomain A cellular fibronectin (EDA-Fn) upon both treatments. Upon RA treatment both Tn polypeptides were also found in extracellular matrix preparations of the differentiated cells. A diffuse extracellular Tn immunoreactivity and a distinct cytoplasmic reaction were seen in differentiated cells especially after exposure to monensin to inhibit cellular secretion. Instead, immunoprecipitation experiments suggested that laminin was synthesized by the cells but was not upregulated upon differentiation. Experiments with purified Tn, used to coat the culture substratum, demonstrated that the undifferentiated cells were unable to adhere or spread on Tn but rapidly acquired the spreading capacity upon differentiation with the inducing agents. In immunofluorescence and immunoblotting the undifferentiated cells presented only a faint heterogenous reaction for beta1 integrin (Int) subunit, whereas cells exposed to RA presented a strong reaction for the Int alpha1 and beta1 subunits, hence suggestive of Int alpha1beta1, and for Int alpha(v) subunit. Cells exposed to TPA showed an enhanced immunoreaction for Int alpha2 and beta1 subunits, suggestive of Int alpha2beta1, and for Int alpha(v) subunit. Immunoreactivity for Int alpha(v) located to distinct punctate plaques in the differentiated cells after both inducing agents. The results suggest that Tn is produced by cultured neuronally differentiating cells, and it is accompanied by the acquitance of an adhesion receptor for Tn.

Expression of laminin in renal-cell carcinomas, renal-cell carcinoma cell lines and xenografts in nude mice.

We studied the expression of laminin (Ln) chains (alpha1-alpha3, beta1-beta3, gamma1) in human renal-cell carcinomas (RCC), papillary renal neoplasms (PRN) and oncocytomas, in RCC cell lines and their xenografts. In RCCs the basement membranes (BM) showed immunoreactivity for chains of Ln-1 (alpha1-beta1-gamma1). Only in well-differentiated RCCs could vessel BMs be distinguished from those of carcinoma cell islets. RCCs and oncocytomas also exhibited an abundant immunoreactivity for Ln beta2 chain in both vessel and tumor cell BMs, while Ln alpha2 chain was not seen in any renal tumors. In distinction from RCCs, PRNs presented a strong BM immunoreactivity for Ln alpha3 and beta3 chains and for Ln-5, as well as lack of Ln beta2 chain. A more variable reactivity for Ln-5 was seen in oncocytomas. As PRNs and oncocytomas have been suggested to originate from collecting ducts, it is notable that in normal human kidney, we could detect immunoreactivity for Ln-5 and its chains only in BM of the tubules of the loop of Henle. In immunoprecipitation experiments, an abundant production of Ln-1, but not of Ln-5, was seen in cultured RCC cells, while in xenografts of the same cells BM-confined immunoreactivity for both Ln-1 and Ln-5 was seen. Ln beta2 chain was produced by 2 of the 4 RCC cell lines in culture but was found only in 1 of the xenografted tumors.

Transforming growth factor-beta regulates the expression of fibronectin and tenascin in BEAS 2B human bronchial epithelial cells.

We used monoclonal antibodies to study expression and extracellular matrix (ECM) incorporation of tenascin (Tn) and isoforms of fibronectin (Fn) in BEAS 2B immortalized human bronchial epithelial cells and the regulation of their synthesis by transforming growth factor (TGF)-beta 1 and -beta 2. In immunofluorescence microscopy, the control cells appeared negative for Tn. Extradomain A (EDA)-Fn was mainly seen in association with ECM fibers and, in a few cells, in an intracellular location. Immunoreactivity for oncofetal (onc)-Fn and extradomain B (EDB)-Fn was only seen in a few cells. In TGF-beta 1- and -beta 2-treated cells, a greatly enhanced immunostaining for Tn and three isoforms of Fn was seen both as to the number of positive cells and to the amount of immunoreactive material around them. In Western blotting of the untreated cells, EDA-Fn and onc-Fn were detected in the cell-free ECM and in the culture medium, whereas EDB-Fn was not detectable. An enhanced secretion and deposition of both EDA-Fn and onc-Fn and also secretion of EDB-Fn was seen upon treatment with TGF-beta s. In TGF-beta-treated cells, Tn was found exclusively in the ECM and not in the culture medium as shown by Western blotting of cell-free ECM and culture medium, respectively. Accentuation of tenascin staining in TGF-beta-treated cells was due to a greatly enhanced production of M(r) 280,000 and M(r) 190,000 isoforms of Tn.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of fibronectin and tenascin production in human bronchial epithelial cells by inflammatory cytokines in vitro.

Fibronectin (Fn) and tenascin (Tn) are two major extracellular matrix glycoproteins participating in tissue morphogenesis and repair. The regulation of their synthesis and deposition during airway inflammation and their possible contribution in asthma are poorly understood. In this study, modulation of Fn and Tn production was investigated in transformed human bronchial epithelial cells in culture. The cells were treated with interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), a combination of these cytokines, interleukins 3 and 6 (IL-3 and IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), and a combination of IL-3 and IL-6 for 48 h. Immunofluorescence and immunoblotting methods with monoclonal antibodies to Fn and Tn antibodies suggested the production of some Fn and Tn in the untreated cells. Fn was minimally induced in response to IFN-gamma and TNF-alpha, when compared with the untreated cells, whereas TNF-alpha and especially the IFN-gamma plus TNF-alpha combination resulted in a prominent Tn induction. Interleukins and GM-CSF did not induce Fn or Tn in any case. These results show that human bronchial epithelial cells are capable of producing Fn and Tn. The modulation of Fn and Tn may have an important impact on the pathology of epithelial cells during airway inflammation in vivo.

Primary and immortalized (BEAS 2B) human bronchial epithelial cells have significant antioxidative capacity in vitro.

Antioxidant enzymes located in the bronchial epithelium can be expected to be important in protecting these cells against both endogenous and exogenous oxidants. In this study, human bronchial epithelial cells were isolated and cultured from specimens obtained from donors for lung transplantation. The levels and relative importance of different antioxidant enzymes were also assessed using an immortalized human bronchial epithelial cell line (BEAS 2B cells). Immunocytochemical studies showed a similar pattern of intracellular localization with the moderate degrees of labeling for Mn superoxide dismutase (SOD), CuZn SOD, and catalase in freshly isolated bronchial epithelial cells, bronchial epithelial cells in primary culture, and BEAS 2B cells. CuZn SOD and catalase decreased in labeling density whereas Mn SOD was unchanged when bronchial epithelial cells were placed in primary cultures. In contrast, Mn SOD and catalase were decreased in BEAS 2B cells compared with primary cultures. Although Mn SOD was low in BEAS 2B cells, it could be significantly induced by tumor necrosis factor treatment. Biochemical analysis showed remarkably similar catalase and glutathione reductase activities in primary cultured epithelial cells and BEAS 2B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoforms of cellular fibronectin and tenascin in amniotic fluid.

Amniotic fluid (AF) obtained from second trimester pregnancies presented extradomain (ED) A, B and an oncofetal (onc-f) domain containing isoforms of cellular fibronectin (cFn) in Western blotting of gelatin-bound polypeptides and directly of AF. Western blotting after sequential immunoprecipitation suggested at least three Fn molecules: one containing EDA and the onc-f domain and another minor component distinctly containing all the domains, and a third one only containing EDA. The immunoblotting results for EDA-cFn and onc-f-cFn were closely similar to that for total Fn, whereas in plasma samples of normal and pregnant women only traces of EDA-cFn and onc-f-cFn, but no EDB-cFn, were found. Western blotting of AF also indicated the presence of three isoforms of tenascin (Tn), M(r) 190,000 and 280,000 polypeptides earlier found in many cells, and a M(r) 200,000 polypeptide, novel for AF and not present in plasma. The results suggest a novel extracellular matrix polypeptide composition for AF.

Production and characterization of monoclonal antibodies specific for different epitopes of human tenascin.

We have obtained and characterized 11 monoclonal antibodies (mAbs) specific for different domains of human tenascin (TN). Five of these mAbs reacted with epitopes contained in the TN area that undergoes alternative splicing and are thus able to recognize specific TN isoforms. These mAbs are a useful tool to study the expression and distribution of TN and its different isoforms in normal and pathological tissues.

Tenascin immunoreactivity in lung tumors.

The distribution of tenascin immunoreactivity was analyzed in nonneoplastic lung tissue, benign lung tumors, and different types of lung carcinomas. In nonneoplastic lung tissue, tenascin could be observed in the basement membranes of the bronchial epithelium and endothelial cells, smooth muscle cells, and bronchial cartilage. Strong tenascin immunoreactivity was seen in the stroma of all the carcinomas of various histologic types. The staining intensity was stronger in the stroma of squamous cell carcinomas than in the stroma of the other types of lung carcinomas. In 10 of 27 squamous cell carcinomas, a granular intracytoplasmic reactivity could also be observed in a subpopulation of tumor cells. Similar intracytoplasmic reactivity was observed in 2 of 27 adenocarcinomas and in both adenosquamous carcinomas. In other types of lung tumors, individual cells did not have intracytoplasmic tenascin, except for one case of leiomyoma, which showed a weak, linear, intracytoplasmic tenascin reactivity. In lung hamartomas, tenascin could be seen in the cartilaginous component of the tumor and in the areas of basement membranes of the bronchial epithelium. In the carcinoid tumors, the stroma displayed a faint positivity for tenascin. These results show that tenascin is widely expressed in the stroma of lung carcinomas. A proportion of lung carcinomas also expressed intracytoplasmic tenascin immunoreactivity, suggesting that tumor cells may be able to synthesize tenascin. In the lung, tenascin positivity is not, however, restricted to malignant neoplasms, as evidenced by the presence of tenascin in nonneoplastic lung parenchyma and in some benign lung tumors.

Human amnion epithelial cells assemble tenascins and three fibronectin isoforms in the extracellular matrix.

Monoclonal antibodies (MAb) were used to show that cultured human amnion epithelial (HuA) cells produce tenascins (Tn) and isoforms of cellular fibronectin (cFn). Tn polypeptides of M(r) 280,000 and 190,000, assembled into extracellular matrix (ECM) but not secreted into the culture medium by HuA cells, were electrophoretically similar to those produced by human fibroblasts as revealed with domain-specific MAbs. The results suggested that most Fn produced by HuA cells contained the extradomain (ED) A and an oncofetal domain but only a minor fraction EDB. In immunofluorescence Tn and Fn were seen in different cytoplasmic granules upon monensin-induced intracellular accumulation. Tn appeared to be deposited in the ECM in colocalization with Fn but distinctly slower. The present results show that cultured normal human epithelial cells synthesize Tn and three isoforms of cFn and secrete them by using different cytoplasmic pathways.

Enhanced tenascin expression in cervical and vulvar koilocytotic lesions.

Tenascin is an extracellular matrix glycoprotein that is widely expressed during embryogenesis. In adults, it is restricted to select sites, including certain epithelial-stromal interfaces, but is notably enhanced in active inflammatory-reactive processes and in the stroma of many neoplasms. The authors immunostained with the monoclonal antibody (MAb) 100EB2 cryosections of vulvar and cervical biopsies displaying convincing koilocytosis with variable degrees of hyperplasia-dysplasia; in situ carcinomas were included. The presence of human papillomaviruses (HPV) 6, 11, 16, 18, 31, or 33 was confirmed by in situ hybridization in a subset of cases. Findings were compared with normal controls. The study was extended with the MAb 143DB7 that reacted with tenascin in paraffin sections. In normal samples, tenascin immunoreaction appeared as a delicate, continuous rim, in the immediate vicinity of the laminin staining; in parakeratotic areas, the rim was thicker. In foci of hyperplastic-dysplastic epithelium with or without koilocytosis, a distinct increase in tenascin staining was noted; enhanced tenascin often paralleled increasing hyperplasia and dysplasia. In most cervical and vulvar carcinomas in situ, the reactions were intense and extended deeply and raggedly into the underlying stroma. Tenascin was selectively enhanced in the endocervical stroma around inflammed or metaplastic glands. The authors conclude that tenascin is increased in HPV infection associated with epithelial proliferation. Enhancement was most consistently strong and extensive in in situ carcinomas, suggesting a correlation with active phases of epithelial growth and stromal remodeling.

A new monoclonal antibody against the GABA-protein conjugate shows immunoreactivity in sensory neurons of the rat.

A monoclonal antibody, 115AD5, was raised against GABA coupled to bovine serum albumin. The monoclonal antibody 115AD5 also reacted with other GABA-protein conjugates. The specificity of the monoclonal antibody was corroborated by enzyme-linked immunoassay, dot-immunobinding experiments and immunostaining of rat cerebellum sections. The monoclonal antibody 115AD5 could successfully be applied on Vibratome and cryostat sections using either indirect immunofluorescence or peroxidase techniques. In rat cerebellar cortex the monoclonal antibody 115AD5 gave an intense immunoreaction in stellate cells, in Golgi neurons, and in basket cells and their processes around Purkinje cell bodies. Purkinje cell dendrites showed GABA immunoreactivity while the cell bodies were non-reactive or only weakly reactive. There was labelling in some nuclei of Purkinje cells. GABA immunoreactivity was also found in dot-like structures in the granular layer. A large population of sensory neurons in rat thoracic and lumbar spinal dorsal root ganglia presented an intense immunoreactivity for the monoclonal antibody 115AD5. Nerve bundles immunoreactive for GABA were also seen in these ganglia. In the trigeminal ganglion, a major population of sensory neurons and some of their processes presented immunoreactivity for GABA. In the sensory nodose ganglion of the vagus nerve, many neuronal cell bodies and some fibres were immunoreactive for GABA. Ligation of the vagus nerve caudal to the ganglion resulted in an increased GABA immunoreactivity in neuronal somata of the ganglion, as well as in nerve fibres on the ganglionic side of the ligature. The present results suggest that in the rat, a population of sensory neurons in thoracic and lumbar spinal dorsal root ganglia, as well as in the trigeminal and nodose ganglia contain GABA. The presence of GABA immunoreactivity in these neurons raises the possibility of a neurotransmitter or modulator role.