Colonic left-side increase of eosinophils: a clue to drug-related colitis in adults.

BACKGROUND: The colon shows frequent eosinophilic infiltration in allergic proctocolitis of infants, whereas in adults, eosinophilic infiltration of the colon is less defined and may be found in different conditions including drug-induced colitis, even though the pathological findings are often inconsistent. AIM: To quantify eosinophils in the mucosa of normal controls and to compare them with those of patients with abdominal symptoms related to 'drug colitis'. METHODS: Mucosal biopsies were obtained during colonoscopy in 15 controls and in 27 patients with abdominal symptoms, a history of probable 'drug-related colitis' and without obvious causes of eosinophilia. RESULTS: The drugs related to the patient symptoms were nonsteroidal anti-inflammatory drugs (70%), antiplatelet agents (19%) and oestroprogestinic agents (11%). Colonoscopy was normal in 30% of patients and abnormal in 70%. Histology showed low content of inflammatory cells and normal crypt architecture in-patients with endoscopy similar to inflammatory bowel diseases. The eosinophil score was significantly higher in the left side of the colon in the patient group compared with controls. CONCLUSIONS: The finding of an increased eosinophil count limited to the left (descending and sigmoid) colon is an important clue towards a diagnosis of drug-related colitis.

Rectal bleeding by Dieulafoy-like lesion: successful endoscopic treatment.

A 81-year old woman affected by chronic renal failure, non insulin-dependent diabetes mellitus (NIDM) and hypertension, had an severe anemia massive hematochezia. The colonoscopy could not localize the bleeding site except some blood spots in the rectum. The patient was readmitted after 1 month with hypovolemic shock by massive hematochezia and required several blood transfusions. The endoscopic examination showed an important arterial bleeding treated successfully with epinephrine and bipolar elettro-coagulation (BICAP). We suggested that the patient presented a Dieulafoy-like lesion; this is an uncommon gastrointestinal cause of bleeding due to a defect of a submucosal artery without evidence of atherosclerosis or vasculitis. Both chronic renal failure and age could be considered as predisponent factors in this patient. Hematochezia is the most important sign and is often complicated by haemorrhagic shock. The diagnosis was delayed due to the difficulty in localizing the bleeding site; moreover, the patient needed several blood transfusions. The arteriographic diagnosis associated to endoscopic treatment by epinephrine and BICAP enabled a successful therapy.

Diazabicyclononanones, a potent class of kappa opioid analgesics.

The 1,5-dimethyl 3,7-diaza-3,7-dimethyl-9-oxo-2,4-di-2-pyridine-bicyclo[3.3.1]nonane-1,5-dicarboxylate, HZ2, has a high and selective affinity for the kappa opioid receptor and an antinociceptive activity comparable to morphine. In addition, it is characterized by a long duration of action and a high oral bioavailability. QSAR studies within series of kappa agonists revealed a chair-boat conformation of a double protonated HZ2 characterized by an almost parallel orientation of the C9 carbonyl group and the N7-H group and at least one aromatic ring to be the pharmacophoric arrangement. Structural variations showed that the pyridine rings in 2 and 4 position can be replaced with p-methoxy-, m-hydroxy- and m-fluoro-substituted phenyl rings. However, all other substituents have to be kept the same for a high affinity to the kappa receptor.

Differential tissue expression of epitopes of the tetraspanin CD151 recognised by monoclonal antibodies.

CD151, a member of the tetraspanin family of cell membrane proteins, is widely expressed in epithelial, endothelial and muscle cells as well as platelets and megakaryocytes. Several monoclonal antibodies recognising CD151 in transfected cells and immunoprecipitating typical bands of 28 and 32 kDa from cell lysates have been produced. Surprisingly, these antibodies show different patterns of staining on tissue sections and on haemopoietic cells. Here we show that these differences are at least in part due to masking of certain epitopes in integrin/CD151 complexes. These data have important implications for the use of monoclonal antibodies in studies of the distribution and function of CD151. Of six monoclonal antibodies from four laboratories, 11B1 was found to be the most reliable for detection of CD151 in different cell and tissue contexts.

Black esophagus: should it be considered an unfavorable prognostic factor?

Black esophagus is a rare condition, reported for the first time in 1990. It is always noted in severely compromised patients. The diagnosis is possible using endoscopy. An esophageal ischemic injury should be considered. It is important that a differential diagnosis is made with consideration of other necrotic conditions of the esophagus. Only supportive treatment and the improvement of the associated disease appear possible.

Synthesis and opioid receptor affinity of a series of 2, 4-diaryl-substituted 3,7-diazabicylononanones.

3,7-Diazabicyclo[3.3.1]nonan-9-ones having aryl rings in positions 2 and 4 with systematically varied substituents were synthesized using a double Mannich procedure. Radioligand binding assays were performed to measure the affinity of the compounds to the mu-, delta-, and kappa-opioid receptors. The affinity of all 2, 4-diphenyl-substituted 3,7-diazabicyclo[3.3.1]nonan-9-ones to the mu- and delta-receptors was found to be low. In contrast, with exception of the nitro- and cyanophenyl-substituted compounds, most of the diazabicycles showed considerable affinity for the kappa-receptor. In particular, the m-fluoro-, p-methoxy-, and m-hydroxy-substituted compounds have an affinity in the submicromolar range. Due to solubility problems in aqueous media, salts of HZ2 were synthesized. The methiodide shows high kappa-affinity and may, thus, be a promising candidate for development of a peripheral kappa-agonist, e.g. for use in the case of rheumatoid arthritis.

Effects of mutant c-kit in early myeloid cells.

Activating mutations in c-Kit, the receptor for Stem Cell Factor (SCF), have been identified in dysplasias and leukaemias of the mast cell lineage and have been shown to contribute to transformation in model systems. Early myeloid cells also normally express c-Kit and their survival, proliferation and differentiation is promoted by SCF. It might therefore be expected that c-Kit mutations could also be involved in some acute and/or chronic myeloid leukaemias. We have found that mutant c-Kit (and normal c-Kit in the presence of SCF) provides a strong differentiation stimulus in normal and immortalised murine early myeloid cells. Since maturation of haemopoietic cells, with the exception of mast cells, results in down-regulation of c-Kit expression, the transforming effects of mutant receptor may be self-limiting in most lineages. This is consistent with the observation that multipotential progenitor cells from some patients with systemic mastocytosis express mutant c-Kit. However, c-Kit mutations have been observed in a few cases of myelodysplastic syndromes or AML without mast cell features. Oncogenesis involves multiple genetic changes and the phenotype of malignant haemopoietic cells expressing mutant c-Kit may be influenced by co-oncogenic events. For example mutations blocking the differentiative effect of mutant c-Kit might result in AML rather than mastocytosis. Thus the extent to which c-Kit mutations contribute to malignancies of early myeloid phenotype remains unknown, and resolution of this issue is complicated by the heterogeneity of this family of diseases.

Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts.

Alternate splicing of mRNA encoding c-KIT results in isoforms which differ in the presence or absence of four amino acids (GNNK) in the juxtamembrane region of the extracellular domain of the receptor. In this study we show that these isoforms of human c-KIT, expressed at similar levels in NIH3T3 cells, display differential effects on various attributes of transformation. The GNNK- isoform strongly promoted anchorage independent growth (colony formation in semi-solid medium), loss of contact inhibition (focus formation), and led to tumorigenicity in nude mice. In contrast, the GNNK+ isoform elicited colony formation but relatively poor focus formation and no tumorigenicity. Saturation binding analysis indicated that the isoforms do not differ significantly in their affinity for the KIT ligand, Steel Factor (SLF). Negligible ligand-independent receptor phosphorylation was observed in either case but, after ligand stimulation, the GNNK- isoform displayed more rapid and extensive tyrosine autophosphorylation and faster internalization. Both isoforms recruited the p85 subunit of phosphatidylinositol 3-kinase and led to similar phosphorylation of its downstream effector c-Akt, but the GNNK- isoform gave rise to more MAP kinase phosphorylation. Thus the c-KIT isoforms display different signalling characteristics and have different transforming activity in NIH3T3 cells.

Effects of mutant c-Kit in early myeloid cells.

Activating mutations in c-Kit, the receptor for Stem Cell Factor (SCF), have been identified in dysplasias and leukaemias of the mast cell lineage and have been shown to contribute to transformation in model systems. Early myeloid cells also normally express c-Kit and their survival, proliferation and differentiation is promoted by SCE It might therefore be expected that c-Kit mutations could also be involved in some acute and/or chronic myeloid leukaemias. We have found that mutant c-Kit (and normal c-Kit in the presence of SCF) provides a strong differentiation stimulus in normal and immortalised murine early myeloid cells. Since maturation of haemopoietic cells, with the exception of mast cells, results in down-regulation of c-Kit expression, the transforming effects of mutant receptor may be self-limiting in most lineages. This is consistent with the observation that multipotential progenitor cells from some patients with systemic mastocytosis express mutant c-Kit. However, c-Kit mutations have been observed in a few cases of myelodysplastic syndromes or AML without mast cell features. Oncogenesis involves multiple genetic changes and the phenotype of malignant haemopoietic cells expressing mutant c-Kit may be influenced by co-oncogenic events. For example mutations blocking the differentiative effect of mutant c-Kit might result in AML rather than mastocytosis. Thus the extent to which c-Kit mutations contribute to malignancies of early myeloid phenotype remains unknown, and resolution of this issue is complicated by the heterogeneity of this family of diseases.

Bisquaternary ligands of the common allosteric site of M2 acetylcholine receptors: search for the minimum essential distances between the pharmacophoric elements.

Structurally diverse molecules, such as alcuronium, gallamine, and tubocurarine as well as W84 and WDUO, are known to interact allosterically with ligand binding to muscarinic M2 acetylcholine receptors. Preliminary molecular modeling studies revealed two positive charges in the middle and two lateral aromatic areas to be essential elements of a high allosteric potency. To find out the optimum distances between these pharmacophoric elements, a systematic variation of the spacer in the series of W84, WDUO, and IWDUO compounds was performed. The allosteric reduction of the rate of dissociation of the antagonist [3H]-N-methylscopolamine from porcine heart M2 receptors served as a test system. The minimal essential distance between the positive charges was found to be 10 A. The length of the peripheral spacers connecting the positive charge and the lateral aromatic moiety appears to depend on the chemical functionality; the peripheral spacers have to be long and flexible enough to position the aromatic skeletons in the spatial neighborhood of the alkane middle chain: in the case of an oxime ether containing peripheral spacer, six atoms are required, and in the case of an alkane chain, four carbon atoms are necessary to adopt the pharmacophoric S-shape conformation.

Transformation of NIH3T3 fibroblasts by the c-Kit receptor tyrosine kinase: effect of receptor density and ligand-requirement.

Ectopic expression of the normal murine receptor tyrosine kinase, c-Kit, in NIH3T3 cells induced many phenotypic changes characteristic of transformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice. Although transformation was largely dependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in the absence of added factor, and this could not be inhibited by neutralising antibodies or by SLF anti-sense mRNA. Clones from factor-independent colonies in semi-solid agar displayed a narrow range of c-Kit surface protein levels (4.3-6.4 x 10(4) receptors/cell) which was relatively high compared with the pool from which they were derived. Analysis of a larger series of random clones derived from adherent cultures expressing different levels of c-Kit demonstrated a positive correlation between SLF-dependent, anchorage-independent growth and c-Kit protein and mRNA expression levels (respectively, Rs = 0.58, P < 0.01; and Rs = 0.53, P < 0.01) with consistent colony formation observed with clones having > 2.5 x 10(4) receptors/cell. Interestingly, two of the three clones expressing the highest levels of c-Kit protein and mRNA produced few or no colonies in the presence or absence of SLF. Sequential overexpression of human c-KIT in NIH3T3 cells using a dihydrofolate reductase (DHFR)-encoding vector and gene co-amplification through methotrexate selection, which resulted in pools expressing up to 1.5 x 10(5) receptors/cell, confirmed that high receptor densities resulted in a decrease in colony numbers. Thus, analysis of clonal and selected populations has indicated that an optimal level of c-Kit is required for transformation of NIH3T3 cells in the presence of SLF, and that some ligand-independent transformation occurs.

Increased expression of c-Kit or its ligand Steel Factor is not a common feature of adult acute myeloid leukaemia.

Cell surface levels of the receptor tyrosine kinase P145(c-kit), the product of the c-kit proto-oncogens, in a panel of 80 primary adult acute myeloid leukaemia (AML) specimens collected at presentation were quantitated by immunofluorescence and flow cytometry, and compared with levels on CD34+ bone marrow cells from normal donors. Receptor levels on AML blast cells were extremely variable and were similar to, or less than, those on normal stem and progenitor cells. In general P145(c-kit) expression was higher on cells of immature phenotype (FAB M1 and M2). c-kit mRNA was quantitated by ribonuclease protection assay (RPA) and was shown to be correlated with cell surface protein expression (r=0.76; P<0.001). This indicates that ligand-mediated receptor internalisation or other mechanisms of increased protein turnover are not responsible for variations in the level of P145(c-kit) in AML specimens. Quantitative Southern blotting was used to examine c-kit gene copy number in 25 of these specimens and was found to be normal in all but one. Thus we have found little evidence of over-expression of c-kit in adult AML. mRNA for the c-kit ligand, Steel Factor (SLF) was also quantitated by RPA in these specimens. While SLF message was detectable (limit of detection approximately 10(4) copies per 10 microgram total RNA; equivalent to 1 copy per 100 cells) in 19% of cases, these specimens in general contained low levels of c-kit mRNA. Thus, an autocrine cycle involving c-kit and SLF does not appear to be a common feature of AML.

Two-step endoscopic resection of gastric leiomyomas.

Our two-step technique for endoscopic treatment of gastric leiomyomas is illustrated. From January 1979 to June 1991, nine symptomatic patients with sessile leiomyomas of the stomach were treated at the Endoscopy Division of Istituto Nazionale Tumori, Milan. The diagnosis was achieved by means of endoscopic observation of the lesion and, when possible, by ultrasound endoscopy. This new technique consists of first removing superficial portion of the tumor by electrosurgical snare. Second, a cleavage plane is found within the proper muscle layer; the tumor is enucleated as much as possible by tightening the snare around it and creating a pseudo-stalk. No major complication occurred nor were any recurrences observed at 21.8 months in the 7/9 patients treated by endoscopy alone. Endoscopic therapy was performed on an outpatient basis and only large lesions required short hospitalization.

Different epitopes of the CD31 antigen identified by monoclonal antibodies: cell type-specific patterns of expression.

3 mAb-5A2.G5, B2B1 and 2BD4-all of IgG1 isotype were identified as belonging to the CD31 cluster by their binding to transfected murine cell lines expressing the CD31 antigen and by sequential immunoprecipitation experiments. Competitive binding experiments were carried out using the human myelomonocytic cell line RC-2A. mAb B2B1 and 2BD4 did not cross block. mAb 5A2.G5 partly inhibited binding of 2BD4 but not B2B1. Thus the epitopes identified by 5A2.G5 and 2BD4 appear to overlap but to be quite separate from that recognized by B2B1. All 3 antibodies bound to a 130 kDa species on Western blots after nonreducing polyacrylamide gel electrophoresis of platelet lysates. Culture of RC-2A cells in the presence of tunicamycin (after removal of surface antigens by pronase) blocked re-expression of the epitopes recognised by all 3 mAb suggesting that they involve N-linked glycosylation. Furthermore, treatment of platelet lysates with Endoglycosidase F prior to electrophoresis and Western blotting abolished the binding of the mAb but not a rabbit polyclonal antiserum to CD31. Nevertheless, neuraminidase treatment of RC-2A cells failed to affect mAb binding. The antibodies displayed typical properties of CD31 antibodies in that all 3 precipitated at 130 kDa cell surface protein and bound strongly to platelets, monocytes, neutrophils and vascular endothelium. All 3 antibodies were positive on hemopoietic progenitor cells which give rise to colonies in the CFU-C assay. However, differences in binding to peripheral blood lymphocytes and to certain human leukemic cell lines were noted. In particular, mAb 5A2.G5 bound weakly to lymphocytes and to the lymphoid cell line HSB-2 compared with the other 2 antibodies.

The murine monoclonal antibody, 14A2.H1, identifies a novel platelet surface antigen.

A murine monoclonal antibody 14A2.H1, raised against acute myeloid leukaemia cells, identifies a previously undescribed 27 kDa platelet surface glycoprotein which is expressed at low copy number (10(3)/platelet). MAb 14A2.H1 caused aggregation of platelets which was dependent on Fc gamma RII. Binding of the antibody to platelets was not altered by activation by thrombin or phorbol ester. In haemopoietic cell populations the antibody bound to megakaryocytes, monocytes (weakly), several myeloid leukaemic cell lines and fresh myeloid leukaemic blasts from some patients. Lymphocytes, lymphoid cell lines, neutrophils and haemopoietic progenitor cells were negative. Expression of the antigen was not restricted to haemopoietic cells as epithelial cells in tonsillar crypts and endothelial cells were positive.

Expression of the YB5.B8 antigen (c-kit proto-oncogene product) in normal human bone marrow.

The c-kit proto-oncogene product is a member of the family of growth factor receptors with intrinsic tyrosine kinase activity. In the mouse c-kit maps to the W locus, which is known to be of central importance in hematopoiesis. Monoclonal antibody (MoAb) YB5.B8, which was raised against peripheral blood blast cells from a patient with acute myeloid leukemia (AML), was recently shown to bind to the extracellular domain of the c-kit product. This antibody does not bind detectably to normal peripheral blood cells and identifies a sub-group of AML patients with poor prognosis. We have used MoAb YB5.B8 to study the expression of c-kit by normal human bone marrow cells by immunofluorescence and flow cytometry, and to isolate multipotential and erythroid colony-forming cells. In a series of 11 normal adult bone marrow specimens, MoAb YB5.B8 bound to 4.0% +/- 1.8% of the cells in the low-density fraction. Dual-labeling experiments were performed with YB5.B8, and CD33, CD34, and CD10 MoAbs. Three populations of cells binding YB5.B8 could be identified based on their pattern of coexpression of the other markers; ie, YB5.B8+/CD34+/CD33-, YB5.B8+/CD34+/CD33+ and YB5.B8+/CD34+/CD33+. These populations had distinctive two-dimensional light scatter characteristics and are likely to correspond to precursor colony-forming cells, colony-forming cells, and maturing mast cells, respectively. No cells binding both YB5.B8 and an MoAb to the early lymphoid marker CD10 were found, implying that most early lymphoid cells do not express c-kit. MoAbs to the c-kit protein should prove valuable in multimarker studies of human hematopoietic stem and progenitor cells. Definition of a reference range of c-kit expression in normal human bone marrow will provide a sound basis for further studies of this marker in diagnosis and prognosis in AML.

[The endoscopic treatment of gastric adenomas]. / Trattamento endoscopico degli adenomi gastrici.

Endoscopic polypectomy is the treatment of choice in symptomatic polyps of the stomach. From 1974 to 1989, at the Endoscopy Division of National Cancer Institute of Milan, 37 patients underwent endoscopic polypectomy, to remove 55 gastric adenomas. Areas of malignancy were revealed in 3 patients. Two of them, not operable for advanced age and poor general conditions, are controlled endoscopically. Endoscopic polypectomy can be performed on an outpatient basis or with a short period of hospitalization. It represents the only form of therapy in not operable patients. Furthermore this method allows to prevent the malignant transformation of the adenomas, which, as reported in the literature, ranges between 3.4% to 66.5%. Our experience confirms the validity of this technique which is safe and effective.

[Endoscopic polypectomy of the stomach]. / Polipectomia endoscopica nello stomaco.

Until 1970 endoscopy of the digestive tract was the only diagnostic method available. After this date, the introduction of endoscopic polypectomy enabled patients affected by these lesions to be treated, and at the same time considerably reduced their length of stay in hospital. The paper reports the results of 346 gastric polypectomies performed in 187 patients from 1974 to the present.

A monoclonal antibody that inhibits the action of GM-CSF on normal but not leukaemic progenitors.

Monoclonal antibody YB5.B8 was previously shown to inhibit haemopoietic colony formation in response to a complex growth factor supplement in vitro (Cambareri A. C., Ashman L. K., Cole S. R. & Lyons A. B. (1988), Leukemia Res. 12, 929). We now report studies of the effect of the antibody on colony formation by normal human bone marrow cells in response to recombinant human colony-stimulating factors GM-CSF, G-CSF and IL-3. MAb YB5.B8 significantly reduced the yield of colonies of all types examined (granulocyte-macrophage, granulocyte, macrophage and eosinophil) in response to GM-CSF but not to IL-3 or G-CSF. However, MAb YB5.B8 failed to influence the proliferation of the myelomonocytic leukaemia cell line RC-2A in response to GM-CSF, G-CSF or IL-3. Direct binding studies demonstrated the presence of low numbers of receptors for GM-CSF on RC-2A cells, however, the binding of this cytokine was not influenced by co- and/or pre-incubation with MAb YB5.B8. Therefore the antigen identified by YB5.B8 is probably not a receptor for GM-CSF and may indirectly influence the response of normal haemopoietic progenitors to this cytokine.