Anne Christine Johannessen

Fas-Induced Apoptosis Is a Rare Event in Sjögren’s Syndrome

The aim of this study was to perform a controlled in situ analysis on the incidence of apoptosis, investigate the expression of apoptosis-mediating proteins, and determine the frequency of apoptotic CD4+ and CD8+ T cells in Sjögren’s syndrome (SS). The study was extended to patients with atrophy-fibrosis (AF) not related to SS, as well as to a control group. Immunohistochemistry and the terminal deoxynucleotidyl transferase mediated dUTP digoxigenin nick end labeling (TUNEL) method were applied to study the Fas and FasL expression and the incidence of apoptosis in salivary glands (SG) from patients with primary and secondary SS, AF, and controls. These methods were also combined to enable simultaneous detection of apoptotic and CD4+ or CD8+ T cells. Despite abundant expression of Fas and FasL in SS SG, apoptotic cells were not exceeding 1% in the foci of infiltrating mononuclear cells (IMC). Double staining showed that the frequency of apoptosis was low among both CD4+ and CD8+ T cells. Only a few TUNEL+ epithelial cells were found in all patient groups. Fas was expressed predominantly on SS IMC, single SS epithelial cells, and a few normal acinar cells, but not in AF SG. Although FasL was present on SS and AF IMC and epithelial cells, it was rarely detected in normal tissue. Consequently we demonstrate that Fas-induced apoptosis among SS SG is a rare event. Our findings support an earlier hypothesis indicating that IMC seem to be able to escape apoptosis, resulting in foci of inflammatory cells. Notably, however, no obvious correlation can be drawn to previous studies where a high incidence of apoptosis of epithelial cells was proposed as an important mechanism leading to decreased glandular function, which is a hallmark of SS.

In situ characterization of mononuclear cells in human dental periapical inflammatory lesions using monoclonal antibodies

Mononuclear cells in cryostat sections of human dental periapical inflammatory lesions were studied with the aid of murine monoclonal antibodies and with indirect immunofluorescence microscopy. T lymphocytes (OKT3-positive cells) made up a major part of the cells in the infiltrates. They were found mainly in clusters, although single cells were also seen. T helper cells (OKT4) were more numerous than suppressor/cytotoxic T cells (OKT8-positive cells), with a ratio of approximately 2:1. Langerhans cells (OKT6-positive cells) were not demonstrated: only a few scattered HNK 1-positive cells, probably natural killer cells, were detected. A large number of OKM1- and OKIa 1-positive cells were detected in the infiltrates. Their size and number varied considerably in the different areas of the sections. These cells are probably macrophages. Sheets of small OKIa 1-positive cells were also demonstrated, indicating the presence of B lymphocytes or activated T lymphocytes. The results indicate that immune reactions may be of importance in the pathogenesis of periapical inflammatory lesions.

Estimation of the prevalence of primary Sjogren's syndrome in two age-different community-based populations using two sets of classification criteria: the Hordaland Health Study

Objective: To estimate the point prevalence of primary Sjögrens syndrome (pSS) in two populations, aged 40–44 and 71–74 years, using two sets of classification criteria. Methods: The participating individuals were recruited from the Hordaland Health Study (HUSK) conducted during 1997–99. A total of 18 592 individuals born 1953–57 and 3346 individuals born 1925–27 were sent a questionnaire covering various health‐related questions, including four questions about sicca symptoms. Among those answering positive to at least one of the four questions, 99 and 90 individuals born 1953–57 and 1925–27, respectively, were examined further. For diagnosis of pSS two classifications were used, the preliminary European criteria from 1993, and the revised European criteria from 1996. Results: By using the two classification criteria from 1993 and 1996, the point prevalences were 0.44% [95% confidence interval (CI) 0.34–0.57] and 0.22% (95% CI 0.15–0.32), respectively, for the population group born 1953–57. The corresponding estimates were 3.39% (95% CI 2.77–4.14) and 1.40% (95% CI 1.02–1.92) for the population born 1925–27. Conclusion: The point prevalence of pSS was approximately seven times higher in the elderly population aged 71–74 years compared to individuals aged 40–44 years, regardless of the classification criteria used.

Retracted: Comparison of histological grading and large‐scale genomic status (DNA ploidy) as prognostic tools in oral dysplasia

This article has been retracted. See retraction notice [DOI: 10.1002/path.2115].

Oral squamous cell carcinoma is associated with decreased bcl-2/bax expression ratio and increased apoptosis

Expression of bcl-2 and bax and apoptosis were studied in fresh frozen samples of normal oral epithelium (OE, n = 7) and oral squamous cell carcinomas (OSCC, n = 16) by immunohistochemistry and the TUNEL method. In OE, bcl-2 was expressed in both basal (96.6% +/- 2.3% [mean +/- SD]) and suprabasal (91.8% +/- 6.2%) compartments. In OSCC, compared with OE, there was a marked reduction of bcl-2-positive cells in the basal part, and in the central parts of well-differentiated (33.0% +/- 19.7%, P < .001) and moderately differentiated (6.1% +/- 4.6%, P < .001) and also in poorly differentiated (1.9% +/- 0.2%, P < .001) tumors. More cells expressed bax in the suprabasal layer of OE (65.6% +/- 9.9%) and central parts of OSCC than in the basal layer of OE (19.1% +/- 4.1%) and basal parts of OSCC. A higher proportion of cells expressed bax in the central part of well-differentiated OSCC (74.3% +/- 8.2%) than in poorly differentiated OSCC (24.9% +/- 9.7%, P < .001). Apoptotic cell death was more pronounced in OSCC (1.5% +/- 0.9%) than in OE (0.4% +/- 0.1%, P < .05). We conclude that, in OSCC, compared with OE, there is a decreased bcl-2 expression, a lowered bcl-2/bax ratio and increased apoptosis. The expression of bax correlates with histological tumor grading in oral squamous cell carcinoma.

CD40, CD154, Bax and Bcl-2 Expression in Sjögren's Syndrome Salivary Glands: a Putative Anti-Apoptotic Role During its Effector Phases

Sjögrens syndrome (SS) is an autoimmune rheumatic disorder characterized by chronic lymphocytic infiltration and decreased secretion in the salivary glands (SGs). For some time, apoptosis has been suggested to constitute the major mechanism for acinar epithelial destruction during the effector phases; however, this is still controversial. We have recently demonstrated that despite the expression of Fas and FasL, the incidence of apoptosis is not increased in SS patients compared with control individuals. Our aim was therefore to further evaluate the expression of the pro‐ and anti‐apoptotic Bax and Bcl‐2 proteins. CD40 and CD154 expression was also investigated, as CD40 ligation has been suggested to protect cells from Fas‐mediated apoptosis. Immunohistochemical staining was performed on SG tissue from primary and secondary SS patients, a group of patients with non‐SS‐related degenerative changes as well as on healthy control individuals. The frequency of stained cells in the foci of infiltrating mononuclear cells (IMCs) and in the acinar and ductal epithelium was determined. We found the expression of Bcl‐2 but rarely Bax in SS SG IMCs. Bcl‐2 in epithelial cells was sparse, while Bax expression occurred frequently and with no significant difference between the patient groups. CD40 and CD154 expression was high among SS IMCs, while CD40 levels were slightly decreased in SS epithelium compared with controls. Elevated CD154 expression was found in SS epithelium, being significantly increased in the ducts. In conclusion, our study further supports the hypothesis about apoptosis resistance among SS focal IMCs and suggests a putative protective role of CD40 ligation in SS SG epithelium.

Sub-Sets of Cancer Stem Cells Differ Intrinsically in Their Patterns of Oxygen Metabolism

The glycolytic response of hypoxic cells is primarily mediated by the hypoxia inducible factor alpha (HIF-1α) but even in the presence of abundant oxygen tumours typically show high rates of glycolysis. Higher levels of HIF-1α in tumours are associated with a poorer prognosis and up-regulation of markers of epithelial mesenchymal transition (EMT) due to HIF-1α actions. We have recently shown that EMT occurs within the CD44high cancer stem cell (CSC) fraction and that epithelial and EMT CSCs are distinguished by high and low ESA expression, respectively. We here show that hypoxia induces a marked shift of the CSC fraction towards EMT leading to altered cell morphology, an increased proportion of CD44high/ESAlow cells, patterns of gene expression typical of EMT, and enhanced sphere-forming ability. The size of EMT fractions returned to control levels in normoxia indicating a reversible process. Surprisingly, however, even under normoxic conditions a fraction of EMT CSCs was present and maintained high levels of HIF-1α, apparently due to actions of cytokines such as TNFα. Functionally, this EMT CSC fraction showed decreased mitochondrial mass and membrane potential, consumed far less oxygen per cell, and produced markedly reduced levels of reactive oxygen species (ROS). These differences in the patterns of oxygen metabolism of sub-fractions of tumour cells provide an explanation for the general therapeutic resistance of CSCs and for the even greater resistance of EMT CSCs. They also identify potential mechanisms for manipulation of CSCs.

Khat (Catha edulis)-induced apoptosis is inhibited by antagonists of caspase-1 and -8 in human leukaemia cells

Khat chewing is a widespread habit that has a deep-rooted sociocultural tradition in Africa and the Middle East. The biological effects of khat are inadequately investigated and controversial. For the first time, we show that an organic extract of khat induces a selective type of cell death having all morphological and biochemical features of apoptotic cell death. Khat extract was shown to contain the major alkaloid compounds cathinone and cathine. The compounds alone and in combination also induced apoptosis. Khat-induced apoptosis occurred synchronously in various human cell lines (HL-60, NB4, Jurkat) within 8 h of exposure. It was partially reversed after removal of khat and the effect was dependent on de novo protein synthesis, as demonstrated by cotreatment with cycloheximide. The cell death was blocked by the pan-caspase inhibitor Z-VAD-fmk, and also by submicromolar concentrations of Z-YVAD-fmk and Z-IETD-fmk, inhibitors of caspase-1 and -8, respectively. The 50% inhibition constant (IC50) for khat (200 μg ml−1)-induced apoptosis by Z-VAD-fmk, Z-YVAD-fmk and Z-IETD-fmk was 8 × 10−7 M as compared to 2 × 10−8 M and 8 × 10−8 M, respectively. Western blot analysis showed a specific cleavage of procaspase-3 in apoptotic cells, which was inhibited by Z-VAD-fmk. The cell death by khat was more sensitively induced in leukaemia cell lines than in human peripheral blood leukocytes. It is concluded that khat induces a rather swift and sensitive cell death by apoptosis through mechanisms involving activation of caspase-1, -3 and -8.

Decreased expression of bcl-2 in moderate and severe oral epithelia dysplasias

The bcl-2 gene family plays an important role in regulation of apoptosis. We previously reported loss of bcl-2 in peripheries of well and moderately differentiated oral squamous cell carcinoma tumour islands. We aimed to determine bcl-2 and bax gene expression in oral epithelial dysplasias (OED) in relation to apoptosis and proliferation. Samples of normal oral epithelium (OE, n=7), focal epithelial hyperplasia (n=9), mild (n=9), moderate (n=8) and severe (n=18) OED were evaluated by immunohistochemistry, the TUNEL method and in situ hybridisation for bcl-2 and bax mRNA. bcl-2 mRNA and protein were markedly decreased in the basal parts of moderate and severe OED compared with the basal layer of OE (P<0.001) and correlated with a 3-4 fold increase in apoptosis and increased proliferation. bax mRNA and protein were not significantly altered in normal, hyperplastic and dysplastic epithelia. From OE to severe OED, there was an inverse relationship between the bcl-2/bax ratio and apoptosis. Our results indicate that suppression of bcl-2 may have a role in oral tumourigenesis.

Catha edulis (Khat) Induces Cell Death by Apoptosis in Leukemia Cell Lines

Abstract: Khat is the Celastraceus edulis plant, a flowering evergreen tree or large shrub, which grows in the Horn of Africa and southwestern Arabia. Khat use has been associated with development of oral cancer, but its molecular effects remain controversial. This study describes a novel cytotoxic effect of whole khat extract on three leukemia cell lines. Cells were exposed to khat extract and harvested for analysis by fluorescent and electron microscopy, trypan blue exclusion, as well as immunoblotting to characterize the mode of cell death. In a separate series, cells were pretreated with a panel of caspase inhibitors for possible inhibitory effects. Khat induced a rapid cell death effect in HL‐60, Jurkat, and NB4 cells that occurred within 2 h of exposure. The treated cells retained their ability to exclude trypan blue dye, a key feature in the apoptotic process. Exposed cells consistently developed morphological features of manifest apoptosis. Z‐VAD, a pan‐caspase inhibitor, completely inhibited toxic activity for up to 8 h, with partial inhibition by other caspase‐specific agents. Western blot analysis showed specific cleavage of caspase‐3 in khat‐exposed cells. This study shows that khat induces cell death by apoptosis in a process sensitive to inhibition by caspase inhibitors, suggesting that subcellular interactions could be of particular relevance for the biological effects of khat in the cell death process and possibly carcinogenesis.