Paired Immunofluorescence Staining Research Articles

Obituary - In Memoriam Per Brandtzaeg.

Obituary - In Memoriam Per Brandtzaeg.

Immune response to Mycoplasma pulmonis in nasal mucosa is modulated by the normal microbiota.

The impact of commensal bacteria on lymphocyte responses in the upper airways was studied in rat nasal mucosa after infection with the pathogen Mycoplasma pulmonis. Phenotyping was performed in situ by paired immunofluorescence staining in germ-free (GF) and conventional (CV) rats before and 3 wk after the monoinfection. Intraepithelial lymphocytes had expanded significantly in GF (P = 0.02) but not in CV rats. Furthermore, a striking proportional increase of T-cell receptor (TCR)alphabeta(+)CD4(+) cells was observed both in the lamina propria and epithelium of GF (P < 0.01) but not of CV rats. Notably, in contrast to the pre-infection state, both mucosal compartments showed a percentage of TCRalphabeta(+)CD4(+) cells that was significantly higher in GF (P = 0.03-P < 0.01) than in CV rats after the monoinfection. In parallel, both compartments displayed a percentage of TCRalphabeta(+) CD8(+) cells that was decreased in GF (P < 0.01) but not in CV rats. The small fraction of TCRgammadelta(+) T cells observed (< 5%) did not change quantitatively or phenotypically after infection. The size of organized nose-associated lymphoid tissue was, on average, increased 5.2-fold in GF rats versus 2.6-fold in CV rats. Collectively, our results demonstrated that the normal microbiota modulated markedly the nasal immune response elicited by monoinfection with M. pulmonis.

Topical application of calcitriol alters expression of filaggrin but not keratin K1 in mouse epidermis.

Calcitriol (1 alpha,25-dihydroxyvitamin D3) and its analogues are antiproliferative agents which promote epidermal differentiation in vitro, possibly reflecting their modes of action in the treatment of psoriasis. We examined the effect of calcitriol on early and late terminal differentiation in mouse epidermis in vivo using an immunofluorescence assay to detect keratin K1 and filaggrin expression. Pulse labelling with the tymidine analogue 5-bromo-2-deoxyuridine (BrdUrd) was performed by intraperitoneal injection of mice immediately or 16 h after a single topical application of 0.72 nmol calcitriol. The BrdUrd labelling index (LI) and keratin K1 or filaggrin expression of postmitotic cell cohorts were scored by paired immunofluorescence staining for up to 72 h after BrdUrd labelling. Calcitriol induced cell proliferation as shown by a 100% increase in the BrdUrd LI 17 h after application. The onset of keratin K1 expression in the postmitotic period was, however, unchanged in both series after calcitriol treatment. Filaggrin expression appeared earlier after calcitriol treatment than in control epidermis, probably reflecting altered cell kinetics with increased epidermal turnover. The results suggest that calcitriol only influences the later stages of the keratinocyte differentiation programme, possibly secondarily to its hyperproliferative effect.

Application of cantharidin or 12-O-tetradecanoylphorbol-13-acetate on mouse epidermis induces a cell population shift that causes altered keratin distribution

Abstract The tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) causes changes in epidermal protein expression, especially in the major differentiation products keratins K1 and K10. These keratins and filaggrin were studied in a pulse-labelled cell cohort in hairless mouse epidermis stimulated to proliferate by TPA or the hyperplasiogen cantharidin. Cells in DNA synthesis were pulse-labelled by 5-bromo-2-deoxyuridine (BrdU) 16 h after topical application of cantharidin or TPA. The BrdU-labelled cell cohort, the two keratins, and filaggrin were spatially mapped by paired immunofluorescence staining. Both cantharidin- and TPA-treated epidermis displayed altered distributions of K1 and K10 with expression only in the outermost cell layers, but the start of their postreplicative expression parallelled that in normal epidermis (18 h for K1 and 24 h for K10 after the last round of DNA synthesis). Cantharidin- and TPA-induced epidermal hyperplasia showed increased basal cell proliferation, accelerated suprabasal migration, and shortened transit time. Thus, the newly formed hyperplastic epidermis was composed of keratinocytes with a lower mean cellular age than that seen in unperturbed epidermis, which caused altered distribution of K1 and K10. Both hyperplastic and normal epidermis showed filaggrin expression in stratum granulosum; this started earlier in treated (30–36 h) than in untreated (96 h) skin. We concluded that the postmitotic onset of K1 and K10 expression was unaltered in regenerative epidermis, whereas filaggrin expression was considerably accelerated and thus influenced by the cell kinetic changes.

Differential effects of topical retinoic acid application on keratin K1 and filaggrin expression in mouse epidermis

Abstract Retinoic acid (RA) modulates epidermal homeostasis and affects differentiation-associated proteins such as keratin K1 and filaggrin. Because results from in vitro and in vivo studies have been conflicting with respect to RA effects on keratinization, we examined the terminal differentiation of epidermal cell cohorts after RA stimulation in vivo. Pulse-labelling with 5-bromo-2-deoxyuridine (BrdU) was performed by intraperitoneal injection of mice immediately or at 16 h after a single topical application of 100 nmol RA. The cell cohort labelled at the time of RA application consisted of previously unperturbed cells exposed to RA after initiation of S-phase, whereas the cohort labelled 16 h after RA application consisted of cells stimulated into the S-phase by RA. Theses two cohorts of partially synchronized cells were followed for up to 72 h after BrdU labelling. Such labelling combined with keratin K1 or filaggrin expression was scored by paired immunofluorescence staining of skin sections. The onset of keratin K1 expression was unchanged in both series after RA treatment, while filaggrin appeared earlier than in controls. The differential effect of RA on the maturation markers was related to the proliferative activity, the increased cell turnover, and the shortened epidermal transit time. The onset of keratin expression appeared to be regulated before the postmitotic period, whereas filaggrin expression appeared to be regulated during the late phase of the maturation process, thus being influenced by the actual epidermal kinetics and structural alterations. These results suggested that the effect of RA on epidermal differentiation is secondary to its effect on proliferation, as determined by the altered cellular age distribution following regenerative proliferation.

Immunohistochemical Identification of Renibacterium Salmoninarum by Monoclonal Antibodies in Paraffin-Embedded Tissues of Atlantic Salmon (Salmo Salar L.), using Paired Immunoenzyme and Paired Immunofluorescence Techniques

Renibacterium salmoninarum was identified in situ by immunoenzymatic and immunofluorescence techniques in paraffin-embedded tissue specimens collected during a natural outbreak of bacterial kidney disease (BKD) and from an experimental infection in Atlantic salmon (Salmo salar L.). Monoclonal antibodies (MAbs) 4D3 and 2G5 were used in this study, both specific for the 57-58-kD outer membrane protein (p57) of the bacterium. Both MAbs revealed positive staining in ethanol-fixed tissue specimens, but only the epitope identified by MAb 4D3 was formalin resistant. Pretreatment with trypsin did not reestablish the antigenicity for the epitope identified by Mab 2G5. Paired immunoenzymatic staining for identification of the bacterium in sequential incubation steps on ethanol-fixed tissue specimens using an avidin-biotin-peroxidase system was obtained after serial dilution of the Mab (2G5) or the chromagen, amino ethyl carbazole, in the first sequence. Paired immunofluorescence staining with well-balanced color mixing was easily obtained on ethanol-fixed tissue specimens using sequential incubations. Single exposures gave blue (aminomethyl coumarin acetic acid) and green (fluorescein isothiocyanate) fluorescence for MAbs 2G5 and biotinylated 4D3, respectively. Color mixing was revealed as a turquoise staining. Studies on method sensitivity was performed by incorporating a known amount of a protein preparation of p57 into an inert matrix, creating an artificial test substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Do human Peyer's patches contribute to the intestinal intraepithelial gamma/delta T-cell population?

T-cell receptor gamma/delta+) (TcR gamma/delta+) lymphocytes in human Peyer's patches (PP) adjoining ileal mucosa were studied by monoclonal antibodies with paired immunofluorescence staining in situ and by flow-cytometric phenotyping of isolated cells. The proportion of gamma/delta+ T cells in the follicle-associated epithelium outside the M-cell areas (median 4.1%, range 2.2-30.1%) was similar to that in mucosal villous epithelium (median 4.4%, range 0.5-30.5%). Most intraepithelial gamma/delta cells (approximately 90%) expressed CD45R0 but only a few expressed CD8 (< 10%) and none L-selectin; a dominating subset (median 46%) employed the V delta 1/J delta 1 gene product (range 22-100%). The M-cell areas lacked gamma/delta cells but contained clustered CD20+ and CD3+ lymphocytes. The subepithelial PP dome area and interfollicular (T-cell) zones, as well as the mucosal lamina propria, contained very few gamma/delta cells (median 1.7%, range 0.4-8.9%) which were dominated (88-100%) by the V delta 2-encoded subset. Those in the dome area and lamina propria were often (approximately 75%) CD45R0+ (range 44-90%) while very few (approximately 2%) expressed L-selectin (range 0-15%). By contrast, CD45R0 expression on gamma/delta cells in the PP T-cell zones was relatively low (approximately 46%) and that of L-selectin relatively high (approximately 43%). In conclusion, TcR gamma/delta + cells are quite rare in human PP and belong mainly to the V delta 2-encoded subset, thus being different from most intraepithelial gamma/delta cells that probably have another origin. The L-selectin+ fraction of PP gamma/delta cells presumably represent newly recruited 'naive' T lymphocytes while CD45R0+ gamma/delta cells both in PP and lamina propria are probably antigen-primed.

Relation between T cell number and epithelial HLA class II expression quantified by image analysis in normal and inflamed human gastric mucosa.

Epithelial expression of HLA class II determinants and the number of lamina propria and intraepithelial T cells were quantified in gastric body mucosa by means of paired immunofluorescence staining which was subjected to computerised image analysis. In normal mucosa, epithelial HLA-DR expression was virtually absent. A significantly increased expression was seen in simple chronic gastritis, most extensively in the isthmus zone, where a positive reaction was seen in 34% of the epithelial area when the gastritis was of low degree and in 85% when it was of moderate severity. The most extensive HLA-DR expression was found in moderate 'stump gastritis' 28 to 32 years after Billroth II resections. In these patients the epithelial area in the foveolar and isthmus zones showed 83% and 92% positive responses, respectively. The HLA subregion products were expressed in a differential manner (DR greater than DP greater than DQ). The number of both intraepithelial and lamina propria T cells increased significantly with increasing severity of gastritis, and the fraction of putative memory T cells was also raised. Correlation analyses showed a positive relation between the epithelial expression of HLA-DR and the intraepithelial as well as the lamina propria density of T cell. These results suggest a biological link between T cells, aberrant HLA-DR expression, and gastritis, although the pathogenic importance of this relation is unknown. Enhanced epithelial presentation of autoantigens or luminal antigens, or both leading to increased activation of T cells is one possible explanation.

Raised number of jejunal IgG2-producing cells in untreated adult coeliac disease compared with food allergy.

The subclass distribution of IgG-producing immunocytes was studied by two colour immunohistochemistry with monoclonal antibodies in jejunal biopsy specimens from 10 adults with untreated coeliac disease, 11 coeliac disease patients on a gluten free diet, and seven patients with established food allergy. Paired immunofluorescence staining was performed with subclass specific murine monoclonal antibodies in combination with polyclonal rabbit antibody reagent to total IgG; the proportion of cells belonging to each subclass could thereby be determined. The ratio of IgG2 immunocytes was significantly higher (p less than 0.05) in untreated coeliac disease patients (median, 35.2%; range, 26.7-65.2%) than in those on a gluten free diet (median, 7.3%; range, 0-31.9%) or those having food allergy (median, 12.5%; range, 0-36.5%). The disparity in the local IgG2 response between patients with untreated coeliac disease and those with food allergy might be due to differences in the nature of the antigenic stimuli, dissimilar genetic 'make-up' of the subjects, or both.

Distribution of immunoglobulin isotypes including IgA subclasses in adult, juvenile, and rapidly progressive periodontitis

The plasma cell population in gingival biopsies from 3 groups of patients with adult, juvenile, and rapidly progressive periodontitis was characterized with respect to the distribution of individual immunoglobulin isotypes, including IgA subclasses, by paired immunofluorescence staining. The median ratios of IgG:IgA plasma cells in gingival connective tissue from the 3 groups were 2.7 (range 2.0-6.5), 3.0 (1.4-6.2), and 2.0 (1.2-4.0), respectively. Cells staining for intracellular IgM were found in low numbers in all biopsies (range 0.3-6.3% of all plasma cells). No statistically significant differences were observed between the 3 patient groups. In all 3 groups, the IgA plasma cell population was predominantly of the IgA1 isotype. One function of IgA seems to be to dampen inflammatory side-effects of other immune effector systems. The demonstrated predominance of IgA1 plasma cells indicates that the majority of IgA produced locally in gingivae of patients with periodontal diseases is susceptible to the IgA1-specific proteases excreted by important members of the disease-associated subgingival microflora. This may be an important factor in the apparently uncontrolled inflammation and tissue degradation taking place in the marginal periodontium during active periodontal disease.

Subclass distribution of mucosal IgG-producing cells in gastritis.

IgG-mediated immune reactions are probably involved in the maintenance of gastritis and glandular atrophy; the mucosal IgG-subclass pattern may therefore influence the effect of local hypersensitivity mechanisms. In this study the proportions of IgG1-, IgG2-, IgG3-, and IgG4-producing immunocytes were determined by paired immunofluorescence staining in specimens from simple gastritis, gastritis after Billroth II (BII) resection, and gastritis associated with dermatitis herpetiformis (DH). The results were related to histopathological degree of inflammation and atrophy. Generally, IgG1 immunocytes predominated (48-60%) in all types of gastritis. With increasing severity of inflammation, the IgG2-cell proportion was significantly increased from 4-6% to 26-34% in simple and BII gastritis, whereas the ratio of IgG1 immunocytes was correspondingly decreased from 58-69% to 38-43%. In the same types of gastritis the proportion of IgG3 cells was increased in association with severe (35-38%) compared with mild (15-23%) atrophy, whereas the proportion of IgG1 cells was correspondingly decreased. In severe gastritis associated with DH, the proportion of IgG1 cells was relatively high (60%) and that of IgG2 cells relatively low (13%), and severe atrophy did not seem to influence significantly the subclass proportions.

J-chain expression is more prominent in immunoglobulin A2 than in immunoglobulin A1 colonic immunocytes and is decreased in both subclasses associated with inflammatory bowel disease

Paired immunofluorescence staining demonstrated reduced J-chain positivity of both immunoglobulin A1 (IgA1)- and IgA2-producing cells in colonic mucosa from patients with ulcerative colitis and Crohn's colitis compared with controls (p < 0.002). J-chain expression was generally higher in IgA2 than in IgA1 immunocytes. The median proportion in normal mucosa was 100% for IgA2 vs. 88% for IgA1 (p < 0.005); in ulcerative colitis, 69% vs. 46% (p < 0.004); and in Crohn's colitis, 74% vs. 46% (p < 0.004). Taken together with the overall IgA-subclass distribution, however, these results showed that the proportion of J-chain-positive IgA2 cells in the total IgA-cell population was lower for ulcerative colitis (20%) and Crohn's colitis (32%) than for normal mucosa (63%) (p < 0.002). In relation to the total J-chain-positive IgA-cell population, which contributes to the secretory IgA system, an increased proportion (p < 0.002) belonged to IgA1 in ulcerative colitis (61% vs. normal, 27%), whereas IgA2 was reduced (39% vs. normal, 73%). Similar but smaller trends were noted in Crohn's colitis. The disease-associated reduction of J chain might be compensated by the previously reported twofold numeric increase of IgA cells in colitis. Our study, therefore, did not suggest that the secretory IgA-cell system was quantitatively impaired in inflammatory bowel disease.

J chain is expressed by more IgA2- than IgA1-producing cells in colonic mucosa, but it is reduced in both subclasses in inflammatory bowel disease.

We have recently observed a shift from the normal predominance of IgA2- (1) to IgA1- producing cells in colonic mucosa from patients with ulcerative colitis (UC) and Crohn’s colitis (CC) (2). This disease-associated alteration may be of great biological importance in terms of secretory immunity. Only J-chain-containing polymeric immunoglobulins(pIg), such as dimeric IgA (pIgA) and pentameric IgM will bind to secretory component (SC) which functions as a pig transport receptor on secretory epithelial cells (3). In this study the capacity of mucosal IgA1- and IgA2-producing cells for plgA formation was examined by paired immunofluorescence staining for subclass and J chain, both in normal and inflamed colonic tissue specimens.KeywordsUlcerative ColitisColonic MucosaSecretory ComponentSecretory Epithelial CellHeavy Chain Constant RegionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Local immunoglobulin production is different in gastritis associated with dermatitis herpetiformis and simple gastritis.

The degree of inflammation and atrophy in gastric body mucosal specimens (n = 38) from 28 patients with dermatitis herpetiformis (DH) was graded histologically. Immunoglobulin (Ig) producing cells were enumerated by paired immunofluorescence staining in a 500 microns wide section area from the muscularis mucosae to the lumen (mucosal 'tissue unit'). The number of immunocytes of the three main classes (IgA, IgM, and IgG) was significantly raised with increasing degree of gastritis. All three classes were increased in specimens showing atrophy compared with those without atrophy. IgA cells predominated as in simple gastritis, but a striking difference was a marked increase of IgM cells in specimens with the most pronounced DH-associated gastritis. Relative class distribution of immunocytes within different mucosal zones showed that the percentage of IgA cells was significantly higher in the luminal than in the basal zone, whereas the contrary was true for IgG cells. IgM cells did not show any zonal preference. No relation was seen between small bowel and gastric lesions. The disproportionate increase of gastric IgM producing cells in DH might nevertheless reflect seeding of precursor cells of the secretory immune system generated in the proximal small intestine where the local IgM response is relatively pronounced.

Mucosal subclass distribution of immunoglobulin G-producing cells is different in ulcerative colitis and Crohn's disease of the colon

As a marked local immunoglobulin G (IgG) response has previously been found to be the most prominent immunopathological feature of both ulcerative colitis and Crohn's disease, the subclass distribution of colonic IgG-producing immunocytes was examined. This study included tissue specimens from 10 patients with ulcerative colitis and 8 with Crohn's colitis. Paired immunofluorescence staining was performed with subclass-specific murine monoclonal antibodies combined with a rabbit antibody reagent of IgG; the proportion of cells belonging to each subclass could thereby be determined in relation to the total number of mucosal IgG immunocytes. A significantly higher median proportion of IgG1 immunocytes was found in ulcerative colitis (81.3%) than in Crohn's colitis (66.5%). Conversely, the median proportion of IgG2 immunocytes was significantly higher in Crohn's colitis (24.9%) than in ulcerative colitis (9.4%). This disparity in the local IgG subclass response might reflect dissimilar mucosal exposure to mitogenetic or antigenic stimuli or genetically determined immunoregulatory differences in the two categories of patients.

Local IgA subclass alterations in ulcerative colitis and Crohn's disease of the colon.

The subclass distribution of IgA producing cells was determined by paired immunofluorescence staining in colonic specimens from 10 patients with ulcerative colitis and eight with Crohn's disease. Compared with normal colonic mucosa, the percentage of IgA1 immunocytes showed a striking increase in both disorders. The proportion of mucosal IgA1 cells was significantly higher (p less than 0.05) in ulcerative colitis (median, 71.2%) than in Crohn colitis (median, 56.9%). Within each category of specimens no significant differences were noted between luminal and basal mucosal zones. The submucosal proportion of IgA1 cells was, however, significantly higher than the mucosal one in both ulcerative colitis (median, 89.1%; p less than 0.002) and Crohn colitis (median, 91.8%; p less than 0.005). The mucosal shift towards IgA1 production paralleled the magnitude of the submucosal IgA1 cell proportion in individual tissue samples. Taken together with the previously reported dramatic increase of local IgG production (particularly observed in the submucosa and basal parts of the mucosa), our findings indicated that there is an influx and/or proliferation of B cells representing systemic secondary immunity in the lesions of both diseases.

Different subclass distribution of IgA-producing cells in human lymphoid organs and various secretory tissues.

A highly reproducible paired immunofluorescence staining method was used to map the relative distribution of IgA1- and IgA2-producing cells in peripheral lymphoid organs and various secretory tissues. Spleen, peripheral lymph nodes, and tonsils all contained a marked predominance (91 to 95%) of IgA1 immunocytes. However, striking variations were demonstrated among the secretory tissues with regard to the median proportion of IgA1-producing cells: nasal mucosa, 96%; lacrimal glands, 81%; major salivary glands, 66%; mammary glands, 63%; gastric and proximal small intestinal mucosa, 84 to 77%; ileum, 55%; and large bowel, 41%. Thus, IgA2 production is relatively enhanced mainly in the distal gut and in mammary and salivary glands, in that order.

Quantitative distribution of immunoglobulin-producing cells in gastric mucosa: relation to chronic gastritis and glandular atrophy.

Immunoglobulin (Ig)-producing immunocytes were quantified by paired immunofluorescence staining in specimens of gastric antral (n = 52) and body (n = 117) mucosa obtained from 45 patients with various gastrointestinal disorders. Enumerations were carried out in a 500 micron wide zone from the muscularis mucosae to the lumen ('tissue unit'). The specimens were divided into three categories according to the degree of inflammation, and each specimen received a grade for atrophy (0-2). The total number of IgA-, IgM- and IgG-producing cells per tissue unit increased strikingly with increasing degree of inflammation, both in antral and body mucosa. IgA immunocytes predominated (61-91%) in all specimens, but the IgG isotype showed the largest relative increase (four to 17-fold), particularly in the basal part of the mucosa. In this layer of the gastric body the proportion of IgG cells was also significantly raised in association with atrophy, irrespective of degree of inflammation. Locally produced IgG may be of protective significance in terms of internal (or 'second line') defence but may at the same time maintain immunopathological mechanisms contributing to the chronicity of gastritis.

Immunohistochemical localization of DNA adducts in rat liver tissue and phenotypically altered foci during oral administration of 2-acetylaminofluorene.

Histological studies using paired immunofluorescence staining and peroxidase-anti-peroxidase staining were performed on sections of rat livers with an antiserum specific for the 2-acetylaminofluorene (AAF)-DNA adduct N-deoxyguanosin-(8-yl)-aminofluorene (dG-8-AF). This is the predominant adduct in rat liver DNA at 5 (80%) and 28 (100%) days of AAF feeding. Nuclear staining was observed in livers of male Fischer rats fed 0.02% AAF for these time periods, and was not present in livers of animals fed control diet or detected when specific antiserum, first absorbed with the immunogen adduct, was utilized. In addition, nuclear staining was unchanged after incubation with RNase and abolished after incubation with DNase. Adducts were not readily detectable when whole-liver adduct concentrations were less than an average of 10(5) adducts per cell (30-50 fmol/micrograms DNA). The overall pattern of adduct distribution in livers of AAF-fed animals was distinctly non-uniform. A predominance of nuclear staining was found in the periportal areas by both immunofluorescence and immunoperoxidase procedures. In contrast, staining was very weak in the centrilobular areas. When animals were fed AAF for 28 days and control diet subsequently for 7, 14, 21 or 28 days, the overall intensity of the immunohistochemical staining decreased with time on control diet. However, the pattern of localization remained the same as in livers of rats fed AAF for 28 days, with the predominance of adducts being in the periportal areas. In male rats fed 0.02% AAF for 8 weeks, foci positive for gamma-glutamyltranspeptidase (GGT) became apparent, and the nuclei in these areas showed no immunofluorescence, indicating the absence of detectable levels of the dG-8-AF adduct. Twenty adduct-negative areas in the median lobes of three rat livers were positive for GGT, which suggests that loss of ability to form adducts in these regions occurs concomitantly with early phenotypic changes.

Immunohistochemical characterization of cellular immunoglobulins and epithelial marker antigens in Warthin's tumor

Lymphoid and epithelial cell marker studies based on paired immunofluorescence staining were performed on ethanol-fixed specimens from six Warthin's tumors of the parotid gland. A polyclonal pattern of isotype and light-chain expression was demonstrated for immunoglobulin-producing cells and afforded definitive evidence for the reactive nature of B-cell proliferation. The average percentages of IgG, IgA, IgM, IgD, and IgE immunocytes were 48.6, 38.5, 8.9, 3.3, and 0.7, respectively. The percentages of J-chain-positive cells within the first four isotypes were 11.3, 47.0, 67.2, and 64.4. Both features were more typical of immune responses in lymphoid tissues than in exocrine glands. In five of the six specimens, IgE was present in a prominent lacy pattern in some follicular centers, often extending to lymphocyte membranes of the mantle zone. Mast cells positive for IgE were seen in all cases. The two latter features indicate that type 1 hypersensitivity might contribute to the lesion. Parts of the tumor epithelium stained selectively for dimeric IgA and secretory component (SC), signifying secretory capacity. In addition, lactoferrin and carcinoembryonic antigen (CEA) occasionally were present in a narrow cytoplasmic luminal rim. Carcinoembryonic antigen was also seen in papillary epithelial projections. Lysozyme was found in isolated epithelial cells, whereas amylase was completely lacking. Except for the presence of CEA, this pattern of epithelial markers resembled that seen in striated ducts of normal salivary glands.