Eosinophilic colitis.

Eosinophilic colitis is a rare chronic inflammatory bowel condition of unknown etiology. We report a case of cecal volvulus causing obstruction in a patient with eosinophilic colitis. A 48-year-old lady presented with abdominal pain, constipation, and abdominal distension. Clinically and radiologically, she was diagnosed to have cecal volvulus. Preoperative colonoscopic reduction failed. At laparotomy, a right hemicolectomy with primary anastomosis was undertaken. Histology of the resected specimen showed diffuse eosinophilic infiltration suggesting eosinophilic colitis. To the best of our knowledge, this association has been never reported.

Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts.

Acridine orange (AO) is a lysosomotropic weak base, a metachromatic fluorochrome, and a photosensitizer, as well. Living cells that are exposed for a short period of time to this compound at low concentration, and under ordinary culture conditions, accumulate the drug within their acidic vacuolar compartment, giving rise to a mainly red, granular fluoresence upon excitation with blue light. When AO-loaded cells are irradiated with intense blue light, AO soon starts to leak from late endosomes and lysosomes, partially shifting the fluorescence to a green, nuclear and diffuse cytosolic, one. This AO-relocalization is a consequence of photo-oxidation of the lysosomal membranes, which initially results in disruption of their proton-gradients and later, in leakage into the cytosol of a host of hydrolytic enzymes--as was here demonstrated by immunocytochemistry--which are capable of causing cellular damage. Most fibroblasts survived minor photo-oxidation, with a period of reparative autophagocytosis. Severe photo-oxidation, which resulted in severe lysosomal damage, caused cellular necrosis; whereas moderate stress, resulting in only partial lysosomal leakiness lead to apoptosis with TUNEL-positive nuclei and shrunken cytoplasm. The findings of the present study show that photo-oxidative damage to the membranes that surround the acidic vacuolar compartment, is an event that results in release of proteolytic and DNA-fragmenting enzymes into the cytosol, which may induce either necrosis, apoptosis, or reparable sublethal damage, depending on the magnitude of lysosomal rupture. Furthermore, the results strongly suggest that proteases and endonucleases of lysosomal origin may induce apoptosis if relocalized from the acidic vacuolar compartment into the cytosol.

Metallothionein and Fas (CD95) are expressed in squamous cell carcinoma of the tongue.

Metallothionein (MT) is a chelator present in myoepithelial cells, whilst the Fas-receptor (APO-1, CD95) has been described primarily in human T Jurkat cells. 20 cases of carcinoma of the tongue were investigated immunocytochemically with regard to MT, Fas and Bcl-2. In normal oral squamous epithelium, MT is located in the basal/parabasal dividing cells only. In well-differentiated nests of carcinomas, MT is observed almost entirely in peripherally located cells. In situ end-labelling indicates apoptosis in the centre of these nests, but not in the peripheral areas. Less-differentiated areas show more general MT-positivity, but little apoptosis. All 24 tumours are Fas-positive, but normal epithelia are mainly negative (P < 0.0001). Bcl-2 protein was sparse in the tumours compared with MT and Fas (P < 0.0001). We thus suggest that MT, possibly due to its chelating properties, may contribute to delaying cells entering apoptosis, both in normal epithelium near the base and in less-differentiated regions of carcinoma. Moreover, Fas may be present in cells of human malignancies, as well as those of established malignant cell lines.

Necrotizing lymphadenitis of the neck (Kikuchi's disease).

Kikuchi's histiocytic necrotizing lymphadenitis is a benign condition originally described in the Japanese literature in 1972. We here describe the clinicopathological features, including fine needle aspirate and bone marrow biopsy, of a case of Kikuchki's disease, which to our knowledge is the first reported from a Scandinavian country. The histopathological features of the enlarged lymph nodes were documented by multiple small necrotic foci showing karyorrhectic debris and haemorrhage. The necrotic foci were surrounded by a mantle of large histiocytic-like cells with vesicular nuclei and clear cytoplasm. The nodal architecture was almost completely effaced and the node infiltrated by a mixture of lymphoid cells of variable size. Neutrophils, eosinophils, and plasma cells were very few. The fine needle aspirate biopsy showed a mixture of small dark lymphocytes, larger activated lymphocytes, and many histiocytes. The bone marrow biopsy showed normal haematopoiesis but some large cells with phagocytosed leukocytes, i.e. similar to haemophagocytosis. Kikuchi's disease has a predilection for lymph nodes in the neck of young women, and is usually self-limited and subsides in 1 to 4 months. The patient described in this report received no treatment. Within 5 weeks the fever subsided and the lymph nodes diminished in size. One year later the patient is well and free of disease. We emphasize the benign nature of Kikuchi's disease, and that SLE and malignant lymphoma are the majori differential diagnoses.

Expression of low molecular weight cytokeratin proteins in laryngeal dysplasia.

In twenty-three cases of laryngeal dysplasia frozen mucosal strips were examined with four monoclonal and one polyclonal keratin antibody. The expression of specific keratin polypeptides was studied in different degrees of dysplasia with regard to the subunits expressed in normal and carcinomatous laryngeal epithelium in the same patient. An alteration in the expression of the subunits of cytokeratin in favour of low molecular weight polypeptides takes place in the transformation of normal epithelium to squamous cell carcinoma. This alteration seems to occur at an early stage and is present already in mild dysplasia. The results suggest that with a suitable antibody dysplastic laryngeal epithelium can be distinguished from normal epithelium, and also on some cases, mild dysplasia from more severe degrees of dysplasia. CAM 5.2, which identifies lower molecular weight cytokeratin proteins (50, 43 and 38 kD), is such an antibody, and can be a valuable diagnostic aid in the histological interpretation of laryngeal dysplasia.

Increase in CD4+ and CD45RO+ memory T cells in the nasal mucosa of allergic patients.

By means of immunocytochemistry we have investigated subsets of T lymphocytes in frozen sections of nasal mucosa from patients with seasonal allergic rhinitis and healthy control persons. All participants were subjected to time-course provocation during the non-pollen season, and samples were taken during provocation as well as during the natural pollen season. Computerized image analysis was applied for evaluation of the immunostained lymphocytes. CD45RO+ memory T cells outnumbered the remaining leukocyte populations in the mucosa of both allergic patients and controls on all occasions. During the repeat provocation there was no difference in numerical values, with respect to any of the five leukocyte subpopulations studied (CD4, CD8, CD25, CD45RA and CD45RO), between patients and controls. However, during continuous exposure in the pollen season a significant increase in CD4+ cells was observed in allergic patients compared to before provocation (p < 0.05). No changes were observed with respect to CD8+ and CD25+ cells. Similarly, an increase in CD45RO+ memory was found in allergic patients during the pollen season compared to the non-pollen season (p < 0.02). This latter finding was, however, only evident in the patients who did not use nasal corticosteroids. Hence the present investigation has demonstrated an allergen-induced increase in CD4+ and CD45RO+ memory T cells in the mucosa of allergic patients during the pollen season. These events may constitute a cellular basis for local continuous production of certain cytokines, particularly interleukin-4, which is essential for IgE synthesis.

Langerhans cells and subsets of lymphocytes in the nasal mucosa.

Langerhans cells and different lymphocytes were studied in the nasal mucosa of 39 woodwork teachers and a control group of 14 healthy subjects. Ten of the woodwork teachers were sensitized as determined by skin prick test. A panel of different monoclonal antibodies was applied on the frozen nasal mucosal specimens. Intraepithelial CD1-positive dendritic cells were found in all specimens. However, there was no difference between the number of these Langerhans cells found in the study group and the number found in the controls. In every specimen the intraepithelial lymphocyte population was dominated by T lymphocytes, and there were relatively few B cells. Similarly the ratio between CD4- and CD8-positive lymphocytes in the study group and the controls was the same. In all specimens there was a dominance of T suppressor/cytotoxic cells compared with T helper/inducer cells. The study confirms that Langerhans cells are present in normal nasal surface epithelium, and suggests that there is no basic difference in the number of Langerhans cells between healthy persons, persons with nasal complaints, and persons with nasal allergy. The dominance of T lymphocytes in the epithelium may indicate the existence of a local cell-mediated immunity other than that associated with the regulation of IgE.

Endothelial adhesion molecules for nasal-homing T cells in allergy.

During the allergic reaction mucosal T cells are activated and a local increase in numbers occurs. In peripheral blood, a concomitant T cell activation and switch towards memory phenotype appears. E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were studied in nasal mucosal biopsies taken during a time-course provocation study, including patients with seasonal allergic rhinitis and healthy controls. Allergic patients were also studied during the natural pollen season with particular attention to the influence of local corticosteroid treatment. Before provocation allergic patients and controls did not differ concerning the expression of endothelial adhesion molecules. However, the epithelial ICAM-1 expression was increased among allergics (P < 0.05). Repetitive allergen provocation induces an increased endothelial expression of VCAM-1 in allergic patients (P < 0.01). Similarly, VCAM-1 expression was increased during the natural pollen season (P < 0.05). Interestingly, the increased VCAM-1 expression was inhibited by the use of local corticosteroids. The present data demonstrate a putative integrin-VCAM-1 mechanism for selective homing of T memory cells to the allergic nasal mucosa and new in vivo effects of local corticosteroid treatment are demonstrated.

Bcl-2 immunoreactivity in salivary gland neoplasms is unrelated to the expression of mRNA for natural killer cell stimulatory cytokines interleukin (IL)-2 and IL-12.

Certain cytokines are involved in the generation of natural killer (NK) cells and participate in the regulation of the proto-oncogene bcl-2. We aimed to study the mRNA expression of interleukin (IL)-2, IL-4 and IL-5, the composition of the tumour infiltrating lymphocytes (TIL), and the expression of bcl-2 in 14 benign and malignant human parotid tumours. T IL were predominantly composed of T lymphocytes and NK cells. We found evidence for the homing of T cells, and for generation of NK cells in the vicinity of the tumours. mRNA for IL-2 and IL-12, were identified but IL-4 mRNA was not found. The cytokine profiles and the composition of TIL of the two tumour categories were indistinguishable, suggesting that these host-response variables do not explain the differences in biological behaviour of these particular tumours. The results support a shift towards Th 1 (T helper 1) cells and interferon-gamma production, and that IL-12 also in vivo may play an important role in the regulatory interaction between innate resistance and adaptive immunity in tumour diseases. Most infiltrating lymphocytes showed strong expression of bcl-2; an interesting observation with regard to lymphocytic apoptosis in neoplastic diseases. The immunoreactivity for the bcl-2 protein varied considerably between and within tumours, and almost all benign tumours showed strong bcl-2 positively whereas several of the malignant tumours showed weak or absent staining. The variable expression of bcl-2 protein suggests a different susceptibility of tumour cells to apoptosis. The results also indicate that bcl-2 cannot pla a major role as protective agent in the specific apoptotic pathway induced by NK cells.

Nasal messenger RNA expression of interleukins 2, 4, and 5 in patients with allergic rhinitis.

In nasal biopsies from 17 adult patients with seasonal allergic rhinitis and from 10 healthy controls, cytokines were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR). The time-course study during winter included repeated local allergen provocation with subsequent nasal biopsies as well as biopsies taken during pollen season. The RT-PCR for CD44 yielded positive bands in 65 of 71 cases, in which cases mRNA for interleukins 2, 4, and 5 (IL-2, IL-4, and IL-5) were thus investigated by means of seminested PCR. IL-4 mRNA was found almost exclusively in the allergic patients. During provocation a significant increase in IL-4 was noticed compared with controls (p = 0.043). Equally, during the natural pollen season, IL-4 mRNA expression was significantly higher in patients not using nasal corticosteroids compared with those who did (p = 0.011). No differences in IL-2 or IL-5 were observed between the groups. These findings also indicate, together with earlier observations of T-cell activation, a phenotype switch toward T-helper 2 (Th2) cells, and the accumulation (homing) of these T cells in the nasal mucosa, that T cells constitute the main source for IL-4 in the nasal mucosa. Therefore, allergic patients have an increased synthesis of IL-4 when provoked with the allergen, and during natural pollen season this synthesis can be downregulated by corticosteroids. Furthermore, this study exemplifies the versatility of molecular biology in surgical pathology and that even low-copy-number cytokine mRNA can be examined in routinely snap-frozen surgical specimens.

Basaloid squamous cell carcinoma of the maxilla.

The study reports the first case of basaloid squamous cell carcinoma (BSCC) involving both the oral mucosa and the tuberosity area of the maxilla. The tumour showed many histological similarities to cases previously reported, though mitoses were not frequent. The immunoreactivity for cytokeratin, S-100, vimentin, Ki-67, p53, c-erbB-2 and bcl-2 was also investigated. Immunostaining for the bcl-2 protein showed a high extent of positive cells, although only a moderate staining intensity. Staining for c-erbB-2 was negative. The pathological findings and the immunoreactivity may indicate that BSCC is not as high a grade carcinoma as previously suggested. Additional studies are thus clearly needed to confirm or reject this impression.

Oxidant-induced apoptosis: a consequence of lethal lysosomal leak?

When macrophage-like J-774 cells are subjected to limited oxidative stress, such as exposure to hydrogen peroxide in a moderate bolus dose, some of their lysosomes rupture-as here assayed by the acridine orange relocalization test-secondary to intralysosomal, iron-catalysed, oxidative reactions. The resultant leakage into the cytosol of hydrolytic enzymes, such as cathepsin-D (as shown here), may initiate a slow degradation/fragmentation process of an apoptotic type within cells still having intact plasma membranes. In contrast, severe oxidative stress also results in extensive lysosomal rupture but leads to necrosis. The chelation of (normally occurring) intralysosomal low-molecular weight iron, by endocytotic uptake of desferrioxamine, largely prevents oxidative stress-induced apoptosis whereas lysosomal iron-loading, by endocytotic uptake of complexed ferric iron, considerably enhances the process. We conclude that oxidant-mediated and iron-catalysed lysosomal rupture leads to decompartmentalization of lysosomal enzymes which in turn may initiate and promote the apoptotic process.

Apoptosis and phagocytosis of tissue-dwelling eosinophils in sinonasal polyps.

OBJECTIVE: Sinonasal polyps contain numerous tissue-dwelling eosinophils, but the mechanisms causing their accumulation, functional activities, and resolution are largely unknown. STUDY DESIGN: Nasal polyp tissue from 14 patients was evaluated for cellular expression of CD95, CD68, and annexin-V, for the degree of apoptosis, and for phagocytosis of eosinophils. MATERIAL AND METHODS: Histological sections were immunostained as single stains for CD95, CD68, and annexin-V, and as an immunostaining for CD68 combined with a modified Vital New Red staining. The latter staining is specific for eosinophils. Other sections were stained by terminal d-UTP nick end labeling (TUNEL) assay and routinely stained for H&E. Evaluation of the amount of stained cells was performed by counting the average number in 10 randomly chosen high-power fields. The TUNEL positivity was in all cases confirmed with apoptotic morphology. RESULTS: The inflammatory infiltrate consisted of numerous eosinophils but also a considerable amount of lymphocytes, mast cells, and macrophage-like CD68+ cells. CD95 was frequently expressed on eosinophils, on numerous other inflammatory cells, and also on morphologically apoptotic cells. annexin-V-positive eosinophils were not as frequent as CD95+ cells, but numerous annexin-V-positive eosinophils were found. CD68+ cells approximately equalled the number of eosinophils. The number of cells phagocytosing eosinophils varied between polyps. Apoptosis of eosinophils (as evaluated by TUNEL combined with apoptotic morphology) was a common finding in six of the polyps. CONCLUSIONS: Previous in vitro and ex vivo findings of CD95 on eosinophils are now supported by demonstration of CD95 on eosinophils in this in vivo study. This investigation revealed a switch of the membrane-bound phosphatidylserine of apoptotic cells, which is a novel observation. The study has demonstrated apoptosis of tissue-dwelling eosinophils, and that CD68+ macrophage-like cells phagocytose eosinophils within the sinonasal polyps.

RANTES is more prevalent in bacterial than in nonbacterial maxillary sinusitis: and P-selectin is preferentially up-regulated in diseased mucosae.

OBJECTIVE: To investigate the relationship of the clinical appearance, histological characteristics, bacterial culturing, and messenger RNA (mRNA) expression of RANTES, interleukin 6, and interleukin 12, as well as the occurrence of endothelial adhesion molecules, in inflammatory diseased maxillary sinus mucosa in critically ill patients. DESIGN: Prospective case series. SETTING: General intensive care unit and neurosurgical intensive care unit of a tertiary care hospital. SUBJECTS: Seven critically ill patients, nasotracheally intubated or tracheotomized, who received ventilator treatment for more than 7 days and treatment with antibiotics. INTERVENTIONS: Bilateral biopsy specimens of antral mucosa were obtained at sinoscopy. Reverse transcriptase-polymerase chain reaction was used to detect the cytokine mRNAs in situ on paraformaldehyde-fixed tissue, and intercellular adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and P-selectin were analyzed by immunochemistry on frozen sections. Sampling of secretion and tissue from the antra was performed for bacterial culturing. RESULTS: Macroscopic and histological appearance varied and showed moderate to pronounced inflammation in 6 antra. All 4 bacterially infected antra showed mRNA RANTES (P=.005). No correlation was found for interleukin 6 and interleukin 12. Up-regulation of P-selectin in all cases and sparse expression of vascular cell adhesion molecule 1 indicate that the inflammation is chronic but nonallergic in type. CONCLUSION: We find an indication that RANTES is more prevalent in bacterial sinusitis.

Quantitative computerized image analysis of immunostained lymphocytes. A methodological approach.

A methodological approach by computerized image analysis to quantify immunostained objects in histological sections is described. We have investigated antibodies against CD4, CD8, CD20, CD23 and CD25 in frozen sections of human nasal mucosa; however, the methodology of standardization is of general validity. The study was designed particularly to investigate the following points: 1) light intensity, 2) the grey level for counter staining intensity, 3) the grey level threshold value for positive objects, 4) the minimal acceptable size of a positive object, 5) the influence of the brightness of the light on both the number and the area of objects. Furthermore, random sampling and determination of 6) the area per section, and 7) the number of histological sections to be measured per biopsy. Finally, a study of reproducibility of immunostaining intensity was performed. The influence of the different parameters mentioned above was studied and the values (eg. threshold value) for our particular setting of microscope, image analysis equipment, computer software etc, were defined. The method was then tested for intra- and interindividual variation which was found to be less than 5%. Correlation analysis of the reproducibility gave coefficients of correlation of 0.99, both concerning number of immunopositive objects and immunopositive area. We emphasize the importance of a highly standardized methodology if the numeric data obtained from computer assisted image analysis are to be more accurate than semiquantitative assessments by experienced observers. With a thorough standardization as described in this method it is possible to obtain numeric values, and data with low deviations, which are two obvious and important advantages.

Immunohistochemical study of epidermal growth factor receptor in severe dysplasia and carcinoma in situ of the vocal cords.

The presence of epidermal growth factor receptor (EGFR) was studied in laryngeal biopsy specimens from 14 patients, all with severe dysplasia or carcinoma in situ. All 14 cases showed positive immunoreaction for EGFR. In 5 of the specimens there were also areas with moderate dysplasia, 3 of which showed scattered EGFR positive cells, whilst 2 were negative. In 8 biopsy specimens there were areas with mild dysplasia, but none of them showed any positive immunoreaction for EGFR. Thus EGFR is present in laryngeal severe dysplasia and carcinoma in situ, indicating the potential malignant neoplastic nature of these lesions. The immunostaining for EGFR may be used as an aid to differentiate between severe dysplasia and carcinoma in situ (Group III lesions), moderate (Group II lesions) and mild dysplasia (Group I lesions).

Expression of epidermal growth factor receptor in laryngeal dysplasia and carcinoma.

The immunoreactivity of epidermal growth factor receptor (EGFR) was studied in laryngeal biopsy specimens from 24 patients. The study comprised 5 cases of normal laryngeal mucosa, 5 cases of dysplasia, 7 cases of well differentiated squamous cell carcinoma, and 7 cases of poorly to moderately differentiated squamous cell carcinoma. EGFR was in general not expressed in normal and dysplastic epithelia, whilst all carcinomas showed a rather strong positive immunoreactivity. There was no significant difference in staining patterns between the well and poorly to moderately differentiated carcinomas. The results suggest that EGFR constitutes a component of neoplastic, but probably not preneoplastic, laryngeal disease. The study failed to reveal any difference in staining pattern between different types of laryngeal squamous cell carcinoma.

Upper airway problems in industrial workers exposed to oil mist.

Exposure to oil mist used in metal work sometimes gives symptoms from skin and airways. This study was performed to evaluate histological and functional respiratory tract disorders. Six male lathe workers aged 31-64 years exposed to oil mist for 4-29 years were examined and compared with matched controls. The investigation included case history, ENT examination, nasal mucociliary function, routine blood tests, IgE, RAST, X-ray of sinus and lungs and biopsy of the nasal mucosa. The mucociliary test showed no difference between the groups. However, all 6 exposed workers had pathological histology findings in the nasal mucosa including lack of cilia, basal cell hyperplasia, goblet cell hyperplasia, squamous metaplasia and subepithelial hyalinization. The biopsies from the controls were mainly normal. The remainder of the investigations revealed no pathology. The study shows that exposure to oil mist--even below the permitted threshold limit--may cause airway symptoms and histological signs comparable to a premature ageing.

Positive identification in situ of mRNA expression of IL-6, and IL-12, and the chemotactic cytokine RANTES in patients with chronic sinusitis and polypoid disease. Clinical relevance and relation to allergy.

Interleukins 6 (IL-6) and 12 (IL-12), and the chemoattractant chemokine RANTES were studied in ethmoidal mucosa, using reverse transcriptase polymerase chain reaction. The 49 patients had chronic sinusitis or nasal/paranasal polyposis, and some also allergy. To the best of our knowledge, this is the first study that demonstrates RANTES and IL-12 on mRNA level in human sinonasal mucosa in situ. mRNA for IL-6, IL-12 and RANTES were detected in 2, 8 and 6 patients with chronic sinusitis, respectively, and in mucosa from patients with polyposis a positive expression was observed in 4, 14 and 10 cases. There were no statistically significant differences. Analysing the entire group of 49 patients, disregarding type of mucosal disease, the number of patients with positive RANTES was significantly higher than that for IL-6. Similarly, IL-12 positivity was more frequently expressed than IL-6. mRNA for IL-6 was expressed in only 2 of the allergic patients. The cytokine production studied thus seems to be unrelated to the clinically defined entities. There is thus a local production in human diseased sinonasal mucosa of RANTES, as well as of IL-6 and IL-12. The local production of RANTES is an important prerequisite for recruitment and migration of inflammatory cells into the tissue. IL-12 is a co-stimulator of antigen-specific responses of established T helper 1 (Th1) clones, and regulates the responsiveness of the clones to a number of T cell growth factors. The study supports a shift towards Th1 cells in these disease entities.

Allergen-induced changes of B-cell phenotypes in patients with allergic rhinitis.

We investigated sub-populations of B-lymphocytes in nasal mucosa and peripheral blood of 17 patients with seasonal allergic rhinitis (birch pollen) and 10 controls. The study included provocation with allergen during the non-pollen season, during which no participant used medication. Samples were also taken during the pollen season. Subsets of B-cells as expressed by different CD antigens were investigated by immunohistochemistry on frozen sections and by flow cytometry of peripheral blood. Nasal CD23+ B-cells decreased in allergic patients during provocation, indicating that mature virgin CD23+ B-cells switch into a memory B-cell phenotype with loss of CD23 expression. This indicates differentiation towards cells that can represent a local source for IgE synthesis. No decrease was observed during the pollen season when the patients used medication. Serum IgE was significantly higher in allergic patients on all occasions. The observed up-regulation of CD40 expression on peripheral blood B-cells in allergic patients during the pollen season clearly indicate B-cell activation. Furthermore, a relative increase of CD19+ B-cells was observed in peripheral blood during provocation. Upregulation (by IL-4) of CD40 on B-cells which then may be stimulated by gp39 (CD40 ligand) can constitute an early and important event in the IgE-mediated allergic reaction.