Massimo Bugnoli

Distribution of cytoskeletal and contractile proteins in normal and tumour bearing salivary and lacrimal glands

We have evaluated by means of immunocytochemistry the distribution of various cytoskeletal and contractile proteins (cytokeratins, vimentin, desmin and α-smooth muscle actin) in 23 salivary or lacrimal gland primary tumours (15 pleomorphic adenomas and 8 carcinomas in pleomorphic adenoma), one third of which contained areas of normal gland. Normal epithelial luminal cells were stained by cytokeratin antibodies with a general specificity, while myoepithelial cells were selectively stained by a monoclonal antibody (SK2-27) reacting in immunoblots with cytokeratin polypeptides 14, 16 and 17, according to the classification of Moll et al. (1982) and by an antibody directed against α-smooth muscle actin (Skalli et al. 1986). In pleomorphic adenomas, both epithelial and myoepithelial cells displayed typical topographic distributions; moreover, myoepithelial cells showed two distinct cytoskeletal phenotypes. These findings could account in part for the heterogeneity of aspects observed in this tumour. In carcinomas, malignant cells were always positive to cytokeratin antibodies with general specificity and myoepithelial cells were absent as judged by anticytokeratin SK2-27 and anti-α-smooth muscle actin immunostainings. However, interestingly, there was in all cases a strong positivity for α-smooth muscle actin in stromal cells, similarly to what has previously been described for mammary carcinoma (Skalli et al. 1986). Our findings may be useful for the interpretation of the histogenesis of salivary and lacrimal tumour and stromal cells.

Inhibition of Helicobacter pylori urease by omeprazole

Objectives To investigate the effect of the proton pump inhibitor omeprazole on the function of purified urease enzyme of Helicobacter pylori and to examine the in vitro activities of omeprazole against urease-positive and -negative Helicobacter strains. Methods Urease was purified chromatographically from H. pylori. The effect of omeprazole on urease enzyme activity was assayed spectrophotometrically after incubation with urea substrate, phenol red indicator and 3mmol/l phosphate buffer. Minimal inhibitory concentrations of omeprazole against H. pylori and other Helicobacter species were determined using an agar dilution assay. Results Omeprazole was found to inhibit the enzyme activity of purified H. pylori urease in a dose-dependent manner. In vitro growth of both urease-positive and mutant urease-negative H. pyloristrains was equally inhibited by omeprazole. Conclusions Whilst omeprazole competitively inhibits the urease enzyme in vitro, urease enzyme inhibition does not account for the in vitro susceptibility of H. pylori to proton pump inhibitors. Urease inhibition by omeprazole in vivo, however, could increase the effectiveness of antibiotics. It may also affect the reliability of the urease-based tests used to assess H. pylori status.

Serologic IgG recognition of Helicobacter pylori cytotoxin-associated protein, peptic ulcer and gastroduodenal pathology in childhood

Objective To assess seropositivity to Heiicobacter pylori cytotoxin-associated proteins in a group of children with H. pylori gastritis and to correlate seropositivity to their disease characteristics, in a prospective study. Patients and methods Sera were collected from 68 children with H. pylori gastritis and IgG recognition of H. pylori cytotoxin-associated proteins was assessed by western blotting. Endoscopic and histologic findings, severity of antral inflammation, mucus gel layer thickness, symptom severity, serum pepsinogen I and gastrin levels of seropositive and seronegative children were compared. Results Forty-four children (64.7%) had serum IgC antibodies that recognized the 130 kDa cytotoxin-associated protein. In seropositive children, the antrum was more frequently nodular at endoscopy (P < 0.01) and duodenal ulcer was more common (P < 0.05). Gastritis was more frequently active (P < 0.01), inflammatory infiltrate was heavier (P < 0.02) and serum pepsinogen I levels were higher (P < 0.001). All children with duodenal ulcer, but only one out of five with gastric ulcer, were seropositive. Conclusions Infection with H. pylori strains which do or do not express the cytotoxin-associated protein may partly account for the difference in disease severity seen in H. pylori gastritis.

Cytokeratin pattern in normal and pathological bladder urothelium: immunohistochemical investigation using monoclonal antibodies.

Normal bladder urothelium and large spectrum bladder lesions have been investigated by immunohistochemistry with monoclonal antibodies of variable specificity (SK 56-23, a large spectrum antibody; SK 60-61, which reacts with cytokeratin polypeptides no. 8 and 18 of Molls catalogue; SK 2-27, specific for polypeptides no. 14, 16 and 17). The normal urothelial pattern is in agreement with previous reports. In pathological conditions, modified immunostaining has been demonstrated in almost all cases. In detail, the cytoskeletal pattern detected in transitional cell papilloma seems to discriminate between types which are otherwise histologically similar. We also observed a correlation between higher degrees of malignancy and loss of specialization, as demonstrated by the increasing positivity for SK 60-61, which as a rule specifically stains umbrella cells, and SK 2-27, an antibody exclusively detected in cells of the basal layer. These findings indicate that the cytokeratin pattern may constitute a modern new tool for the pathologist in the diagnosis of urothelial proliferative disorders.

Pathogenic Mechanisms of Helicobacter pylori: Production of Cytotoxin

Bacteria associated with mucosal infections of the digestive system generally produce toxins, especially when they cause inflammatory lesions. Illnesses due to thermotolerant campylobacters, to enterohemorrhagic Escherichia coli, and to Clostridium difficileare only some examples. It would be surprising if Helicobacter pylori (HP) did not produce any toxic substances. The difficulty consists in attributing a pathogenic meaning to the toxin, since the range is quite wide of clinical and histological presentation of gastroduodenal inflammatory diseases linked to the presence in the stomach of H. pylori organisms [1]. Johnson and Lior [2] firstly reported the production of heat-labile cytotoxin by 80.6% of 36 HP strains they tested. However, most of our knowledge of the cytotoxigenicity of HP is from Leunk et al. [3] whose work has inspired us in part. They found that about 55% of 201 HP strains isolated in four different parts of the world produced a substance which caused intracellular vacuolization in cells of several lines in vitro, not only in lines generally employed in toxigenicity tests, like Chinese hamster ovary (CHO) cells, Vero cells, and Y-1 cells, but also in human tumoral cells like HeLa, KATO III, and HEp-2, as well as in human embryonic intestinal cells which were the most responsive. They also inferred that the toxin was proteinaceous in nature being heat labile (destroyed at 70 °C for 30 min), protease sensitive, and ammonium-sulfate precipitable. Its molecular weight ought to be higher than 100 kDa since cytotoxic activity could be found only in the retentate of a concentrated broth culture filtrate (CBCF) passed through 100 kDa molecular weight limit ultrafiltration membrane.

Expression of cytokeratin polypeptides in human papillomavirus (HPV) lesions of the uterine cervix: 1. Relationship to grade of CIN and HPV type.

SummaryA series of 74 punch biopsies, derived from 513 women prospectively followed for cervical human papillomavirus (HPV) infections (including HPV-NCIN, HPV-CIN I, HPV-CIN II, and HPV-CIN III lesions), and 43 control cases (consisting of normal epithelia, nonspecific cervicitis, and classical CIN lesions) were analysed for expression of cytokeratin polypeptides using the ABC technique and monoclonal antibodies SK 56–23 (wide-spectrum antibody), SK 60–61 (specific for keratins 8 and 18), and SK 2–27 (detecting keratins 14, 16, and 17). HPV typing was carried out using the in situ hybridization technique with DNA probes for HPV 6, 11, 16, 18, and 31. All layers of the exocervical epithelium were regularly stained with the antibody SK 56–23, and the staining pattern remained unaltered in all cervical lesions studied. In contrast to the normal exocervical epithelium, which remained negative with SK 60–61, positive staining was observed in 3 of 15 cervicitis cases and 6 of 23 classical CIN lesions. Interestingly, the majority (69 of 74, 93.2%) of both HPV-NCIN and HPV-CIN lesions showed positive staining with this antibody either in all layers or in suprabasal cells. Antibody SK 2–27 stained the basal cells of the normal exocervical epithelium with remarkable specificity. In 18 of 19 HPV-NCIN lesions, basal cells could not be stained by SK 2–27 monoclonal, but the suprabasal cells were stained instead. In HPV-CIN, but not in classical CIN, this antibody demonstrated the presence of the epitope typical of the cytokeratins 14, 16, and 17 in all layers of the epithelium, the highest frequency (9 of 12, 75%) being found in HPV 16-induced lesions. These disturbances of cytokeratin patterns in cervical epithelium could be associated with cell transformation by HPV, leading to development of HPV-CIN, and could be specific for this virus. The present data are of interest in assessing the stage of maturation of the squamous cells in progressing cervical HPV infections.

The 130-kDa Vacuolating Cytotoxin-Associated Protein Is a Component of Cytotoxic Helicobacter pylori Organisms

Helicobacter pylori (HP) is the agent of type B gastritis and a cofactor in the development of peptic ulcer and probably also of gastric cancer. Certain HP strains produce a toxin which induces intracellular vacuolization on mammalian cells in vitro. Proteins associated with vacuolating activity (VA) have molecular weights of 130, 95, and 80 kDa and can be detected broth and in concentrated broth culture filtrates (CBCF) of cytotoxic strains [1].

Demonstration of the major cytotoxin-associated protein of Helicobacter pylori in gastric biopsies by Western Blotting.

Helicobacter pylori (HP) strains differ considerably in their virulence characteristics. Only certains organisms, for example, produce a toxic substance which causes intracytoplasmic vacuolization on cells in vitro [2, 3]. Cytotoxic HP strains (CTHP) can also be considered ulcerogenic because they have been isolated with greater frequency from patients with peptic ulcers than from patients with chronic gastritis only [2]. Thus the vacuolating toxin most probably plays a crucial role in the pathogenic mechanism of the gastric mucosa damage.