The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Fenoldopam to prevent renal replacement therapy after cardiac surgery. Design of the FENO-HSR study.

INTRODUCTION: Acute kidney injury requiring renal replacement therapy is a serious complication following cardiac surgery associated with poor clinical outcomes. Until now no drug showed nephroprotective effects. Fenoldopam is a dopamine-1 receptor agonist which seems to be effective in improving postoperative renal function. The aim of this paper is to describe the design of the FENO-HSR study, planned to assess the effect of a continuous infusion of fenoldopam in reducing the need for renal replacement therapy in patients with acute kidney injury after cardiac surgery. METHODS: We're performing a double blind, placebo-controlled multicentre randomized trial in over 20 Italian hospitals. Patients who develop acute renal failure defined as R of RIFLE score following cardiac surgery are randomized to receive a 96-hours continuous infusion of either fenoldopam (0.025-0.3 µg/kg/min) or placebo. RESULTS: The primary endpoint will be the rate of renal replacement therapy. Secondary endpoints will be: mortality, time on mechanical ventilation, length of intensive care unit and hospital stay, peak serum creatinine and the rate of acute renal failure (following the RIFLE score). CONCLUSIONS: This trial is planned to assess if fenoldopam could improve relevant outcomes in patients undergoing cardiac surgery who develop acute renal dysfunction. Results of this double-blind randomized trial could provide important insights to improve the management strategy of patients at high risk for postoperative acute kidney injury.

Interrelationships between "Candidatus Phytoplasma asteris" and its leafhopper vectors (Homoptera: Cicadellidae).

The titer of chrysanthemum yellows phytoplasma (CYP, "Candidatus Phytoplasma asteris") in the three vector species Euscelis incisus Kirschbaum, Euscelidius variegatus Kirschbaum, and Macrosteles quadripunctulatus Kirschbaum (Homoptera: Cicadellidae) was measured after controlled acquisition from infected Chrysanthemum carinatum (Schousboe) (daisy) plants. Phytoplasma DNA was quantified in relation to insect DNA (genome units [GU] of phytoplasma DNA per ng of insect DNA) by using a quantitative real-time polymerase chain reaction (PCR) procedure. The increase in phytoplasma titer recorded in hoppers after they were transferred to plants that were nonhosts for CYP provides definitive evidence for phytoplasma multiplication in leafhoppers. CYP multiplication over time in M. quadripunctulatus was much faster than in E. incisus and E. variegatus. CYP titer was also highest in M. quadripunctulatus, and this was reflected in the latent period in the insect. The mean latent period of CYP in M. quadripunctulatus was 18 d versus 30 d in E. variegatus. M. quadripunctulatus was the most efficient vector, giving 100% transmission for single insects compared with 75-82% for E. incisus or E. variegatus, respectively. By sequential transmission, we analyzed the time course of transmission: E. variegatus were persistently infective for life or until shortly before death. Occasionally, leafhoppers failed to maintain continuity of infectivity even after completion of the latent period. PCR analysis of transmitter and nontransmitter E. variegatus adults showed that some nontransmitters were CYP positive, whereas others were CYP negative. These findings suggest that both midgut and salivary gland barriers play a role in transmission efficiency.

Typing of human papillomavirus DNAs by restriction endonuclease mapping of the PCR products.

The polymerase chain reaction (PCR) for the diagnosis of human papillomavirus (HPV) infections, and in particular for the study of cervical HPV-associated lesions, is used widely. We identified a novel set of universal primers that are able to amplify a fragment spanning the E1 open reading frame (ORF) from different mucosotropic HPV types. A restriction endonuclease digestion of the amplified products is suggested for accurate typing. In particular, AluI digestion of the amplified fragments yields a distinctive fragment pattern for each 'high-risk' (16, 18, 31 and 33) HPV sequence, thus distinguishing them from 'low-risk' (6b and 11) HPV sequences.

Immunohistochemical study on the pattern of 50 kd cytokeratin in psoriasis.

Three biopsies of normal skin and 15 biopsies collected from patients with psoriasis vulgaris were analyzed for the expression of the 50 kd cytokeratin using direct immunofluorescence and ABC technique before and after local treatment with anthralin 0.1% in a petrolatum base, with 0.05% betamethasone dipropionate cream, and finally, after PUVA treatment. Antiserum against the 50 kd anti-cytokeratin reacted with tissue sections of normal skin, staining cells in the basal layer, while the psoriatic skin sections before the various treatments showed a staining concerning the whole thickness of the epidermis. After the various therapies, the 50 kd cytokeratin immunoreactivity was observed only in the basal layer of those psoriatic skin sections that showed complete clinical clearing, while it was observed in the whole thickness of psoriatic patches that did not clear. These data suggest that the normalization of the 50 kd cytokeratin expression pattern can be considered as a marker of clinical remission of psoriasis.

Multianalytic study on cervical HPV lesions.

Nowadays new sophisticated techniques of molecular biology based on the principles of hybridization between nucleic acids, allow a correct diagnosis of genital HPV infection. In the present paper, beside traditional diagnostic methods, we used In Situ Hybridization (ISH) and Polymerase Chain Reaction (PCR) to detect the presence of HPV types 6, 11, 16, 18, 31 and 33. We tested ten patients affected by cervical lesions of high histological atypias associated with HPV, who underwent surgical conization. Types 6 and 11, at low risk of evolution, are less frequent than 31 and 33, at medium grade of evolution, and than 16 and 18 which are at high risk of evolution.

Expression of alpha-smooth-muscle actin in stromal cells of the uterine cervix during epithelial neoplastic changes.

A total of 55 formalin-fixed and paraffin-embedded specimens of normal, inflamed and neoplastic uterine cervix have been studied in order to correlate the epithelial changes with the expression of alpha-smooth-muscle actin in stromal cells. This actin isoform is typical of smooth-muscle cells, but appears also temporarily in fibroblasts during wound healing and permanently during fibrocontractive diseases and stromal reaction to epithelial tumors. While positive stromal cells were absent in normal and inflamed cervix, they accumulated in relation to neoplastic tissues. The number of alpha-smooth-muscle actin positive cells and the intensity of stain were related to the increasing grading of cervical intra-epithelial neoplasia. Our results suggest that alpha-smooth-muscle actin is a marker of stromal-cell reaction to the development of neoplastic lesions. The evaluation of alpha-smooth-muscle actin in stromal cells of the uterine cervix may be a useful adjunct to diagnostic criteria of cervical intra-epithelial neoplasia and may help understanding of the mechanisms of mesenchymal-epithelial interactions during neoplasia.

Cytokeratin pattern in normal and HPV infected oral mucosa in women with genital HPV infections.

The distribution of cytokeratins Nos. 19 (CK 19), 14, 16 and 17 (CK2-27), and 8 and 18 (CK 60-61) in 96 oral mucosal biopsies taken from women with genital HPV infections were studied by immunohistochemistry, using polyclonal antibody CK 19, as well as monoclonal antibodies CK 2-27 and CK 60-61. White staining of the buccal mucosa after acetic acid application, which recently was shown to be affected mostly by smoking and age, could not be explained by differences in cytokeratin pattern. In HPV DNA-positive biopsies, the staining with CK 19 antibody in the basal cell layer was more intense than in HPV DNA-negative biopsies. The staining with CK 2-27 antibody was seen in 76% and 91% of the basal and superficial layers, respectively, even though these low molecular weight cytokeratins should be found mainly from the basal and parabasal cells. CK 60-61 staining was almost similar to that seen recently in normal genital mucosa. When trying to distinguish oral HPV infections from normal mucosa, CK 2-27 and CK 60-61 stainings were of no diagnostic value. The more efficient expression of CK 19 in HPV DNA-positive samples suggests that viral infection might accelerate the production of low molecular weight cytoskeletal protein. This could be interpreted as evidence that HPV might disturb the keratinocyte differentiation in the basal cells. As a result of the present study, CK 19 staining in oral mucosa needs to be further studied in regard to viral infections, because it may help to better understand the interaction between a virus and a host cell.

Detection of S-100b protein in Triton cytoskeletons: an immunocytochemical study on cultured Schwann cells.

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.

Immunolocalization of intermediate filament proteins using antibodies to synthetic peptides.

Synthetic peptides corresponding to amino acid sequences of amino terminal non-alpha helical domains of human cytokeratin 18 and to low molecular weight human neurofilament subunit were used to obtain monospecific antisera. The results of our immunohistochemical investigations confirmed in general the data previously published on the distribution of cytokeratin 18 in human, rat, and calf tissues. The reactivity of the antiserum was abolished after formalin fixation of specimens. Immunolocalization of the neurofilament subunit using our monospecific antiserum was quite variable from species to species in cells of the central and peripheral nervous systems, and also varied as the result of the tissue fixation procedures. In particular, formalin fixation destroyed the immunoreactivity of the recognized epitope. We discuss the advantages and limits of the use of synthetic peptides as immunogens to produce polyclonal antibodies against intermediate filament proteins, with particular attention to the epitope masking phenomena in cytokeratin polypeptides and the phosphorylation of epitopes in neurofilament subunits.

Immunohistochemical localization of filaggrin in benign and malignant lesions of the human oral mucosa.

Filaggrin is a protein normally present in the granular and horny layer of stratified squamous epithelia. We studied the presence of this protein in 83 benign lesions and in 73 cases of malignant epithelial tumours of the oral cavity and investigated its possible role as an immunohistochemical marker. The immunohistochemical technique was based on the P.A.P. method. The results in benign lesions show a distribution of filaggrin similar to that observed in the normal mucosa. By contrast, an irregular distribution of filaggrin is observed in areas of leukoplakia with parakeratosis and in papillomas. In malignant lesions the expression of this protein is closely related to the degree of differentiation of the cellular elements, being positive in more differentiated and negative in anaplastic areas. Therefore in some types of benign lesions filaggrin testifies an alteration of the normal process of keratinization. Filaggrin is more significant in malignant lesions in which its presence if any permits an evaluation of the degree of differentiation of the tumour.

Expression of cytokeratin No. 19 polypeptide in genital papillomavirus lesions.

A series of 23 punch biopsies proved to contain human papillomavirus (HPV) type 16 and with established clinical course (including HPV-NCIN, HPV-CIN I, and HPV-CIN II lesion), and 18 additional biopsies of HPV 6-, 11-, 16- or 18-induced genital lesions were analyzed immunohistochemically for expression of cytokeratin No. 19 polypeptide. An immunoperoxidase-ABC technique was used with a polyclonal antibody raised against a synthetic nonapeptide corresponding to the residues 2-10 of the NH2-end, non-alpha-helical region. This polyclonal cytokeratin No. 19 antibody stained mainly (but not exclusively) the basal cells of the normal exocervical epithelium (heterogeneous pattern). Basal cell staining was intense slightly more frequently in HPV-CIN than HPV-NCIN lesions, i.e., ++ or more in 14/24 (58.3%) versus 8/17 (47.0%), respectively. The difference was more marked in the staining of the superficial cells, 70.8 and 58.8% showing intense expression of cytokeratin No. 19, respectively. In 6 (21.4%) of the 28 HPV 16 lesions, basal cell layer was intensely stained, as contrasted to none of the 13 HPV 6, 11 or 18 lesions. The most distinct feature was the well-defined granular staining pattern of the superficial layer in 8 out of 10 HPV 6/11 lesions, as contrasted to the homogeneous pattern in 24 out of 28 HPV-16-infected lesions. In superficial cells, regressed lesions exhibited intense staining in 9/13 (69.2%), as compared with only 4/10 (40%) of the progressed lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topography-related expression of individual cytokeratins in normal and pathological (non-neoplastic and neoplastic) human oral mucosa.

Recently, regional changes of cytokeratin patterns in human normal non-keratinized or keratinized oral mucosa have been demonstrated and the expression of individual cytokeratin polypeptides in lesions of oral mucosa has been compared with that of normal tissues. In particular, the presence of cytokeratin 19 in the suprabasal cell layers of oral epithelia has been shown to be strongly correlated with premalignancy. In the present study, we describe the results of an immunohistochemical investigation performed using a monoclonal antibody specific for cytokeratin 1 on normal oral mucosa and benign or malignant oral lesions. We show the different distribution of this polypeptide in non-neoplastic lesions from different sites of oral mucosa and describe the presence of cytokeratin 19. Our results are in agreement with the data obtained previously. In the malignant cases we demonstrate that the distribution of the two cytokeratins is characterized by complementary patterns.

Expression of phosphorylated and non-phosphorylated neurofilament subunits and cytokeratins in neuroendocrine lung tumors.

In this study antibodies specific for different intermediate-sized proteins (cytokeratins and neurofilaments) have been tested on a series of neuroendocrine (NE) lung tumors in order to evaluate their diagnostic validity. In particular we used a panel of polyclonal anti-neurofilament 200-kilodalton subunits whose reactivity against phospho-dependent epitopes was known. At least one NF subunit was constantly present and in all cases coexpression of cytokeratins and neurofilaments was confirmed. However, in cases of carcinoid tumor (CT) the results were homogeneous, while the cases of small cell lung carcinoma (SCLC) showed a much wider range of immunostaining. Our investigation confirms the hypothesis that the phosphorylation state is a significant determinant of immunohistochemical properties of neurofilaments. This might explain the large number of negative results obtained in previous investigations on NE tumors. The phosphorylation of neurofilaments may also be considered an indication of the degree of differentiation of the tumor.

Expression of actin isoforms and intermediate filament proteins in childhood orbital rhabdomyosarcomas.

The diagnosis of orbital rhabdomyosarcoma (RMS) in childhood gives rise to several clinical and anatomo-pathological problems. Antibodies recognizing structural proteins and cytoskeletal components have been shown to increase the diagnostic accuracy of different neoplastic lesions. In this study we examined anatomo-clinically and, where possible, by means of immunohistochemistry and electron microscopy, a series of 14 cases of orbital RMS in childhood. In the 12 cases studied by immunohistochemistry, desmin was always present, although showing variable patterns, and alpha-sarcomeric actin was found in 10 cases. alpha-Smooth muscle actin was always absent. The other markers tested (myoglobin, polyclonal actin, vimentin and enolase) proved unreliable for several reasons. We conclude that antibodies against desmin and alpha-sarcomeric actin are useful for the diagnostic definition of RMS. In addition, immunohistochemical analysis supplies data regarding the degree of tumor differentiation and may be applied to monitor radio- and chemotherapy.

[Study of filaggrin in psoriasis]. / Studio della filaggrina nella psoriasi.

Filaggrin is a histidine-rich basic protein that aggregates keratin filaments in fully differentiated cells of the epidermis. Filaggrin is synthesized in the granular cell layer as a high-Mr phosphorylated precursor, profilaggrin, that is processed to form the lower-Mr product present in cornified cells. The catabolism of filaggrin in stratum corneum produces urocanic acid and carboxylic-pyrrolidone acid that, respectively, absorb UV radiations and support cutaneous hydratation. In this study we evaluated by direct-immunofluorescence and by immunoperoxidase staining using rabbit antihuman filaggrin antiserum localization of filaggrin in psoriatic skin and in normal human skin before and after treatment with anthralin 0.1%, betamethasone 0.05% and hydrocolloid dressing. Antiserum against human filaggrin reacted with tissue sections of normal human skin, staining cells in the granular layer and in the stratum corneum, while no staining of human psoriatic skin sections was observed. After treatments, filaggrin resulted present in those psoriatic skin sections that showed complete clinical remission, while it was not observed in psoriatic patches which did not clear. These studies suggest that human skin filaggrin can be considered a marker of clinical remission of psoriasis.

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 Moll's 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.

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

A 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.

Altered expression of filaggrin in human papillomavirus (HPV) lesions of the uterine cervix.

A series of 64 punch biopsies collected from women prospectively followed-up for cervical Human papillomavirus (HPV) infections (with and without CIN), and 38 control biopsies (normal epithelia, and classical CIN) were analysed for expression of filaggrin (a histidine-rich protein constituent of keratohyalin granules) using the ABC technique and polyclonal antibody. HPV typing was completed using the in situ hybridization technique with DNA probes for HPV 6, 11, 16, 18 and 31. Three patterns of filaggrin distribution were differentiated: pattern I, all layers above the basal cells stained positive regularly; pattern II, all layers above the basal cells stained irregularly, and pattern III, scattered superficial cells stained positive. There was a significant difference between HPV-noCIN and HPV-CIN lesions in their filaggrin patterns, pattern I being present in the majority (77.7%) of HPV-noCIN lesions, as contrasted to HPV-CIN lesions, where pattern III was the predominant one (43.5%), followed by pattern II (32.6%). In HPV-CIN as well as in CIN lesions, pattern I was inversely related to the grade of CIN, being entirely absent in HPV-CIN III and CIN III. A significant difference exists between CIN and HPV-CIN lesions, concerning the presence of pattern III (4.3% and 43.5%, respectively, P less than 0.001). The difference was less dramatic with regard to pattern I (30.4% and 21.7%, respectively, P less than 0.05). In the lesions containing HPV 6, 11 or 31 DNA, filaggrin distribution was shown to be more close to that of the normal epithelium (I 36.7%, and II 34.7%), while in the HPV 16 and 18-infected cases, pattern III was the predominant one (46.7%). The assessment of filaggrin pattern in HPV lesions might be of help in evaluating the severity of the disturbance of keratinocyte differentiation induced by the progression of HPV infections.

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 alpha-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 alpha-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-alpha-smooth muscle actin immunostainings. However, interestingly, there was in all cases a strong positivity for alpha-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.