Apmis

ZUBAC, D. P.; BOSTAD, L.; KIHL, B.; EIDE, J.; WENTZEL‐LARSEN, T.; HAUKAAS, S. A.

doi: 10.1111/j.1600-0463.2008.01071.xpmid: 19133004

The frequency of diagnosed and treated organ‐confined renal cell carcinoma is increasing. The prognosis of this group of tumours is difficult to predict. The main purpose of this study was to examine the prognostic significance of microvascular invasion, tumour size and nuclear grade in a complete cohort of 76 consecutive patients with organ‐confined clear cell renal cell carcinoma treated with radical nephrectomy. Patient ages ranged from 39 to 88 years (mean 66 years). Median follow‐up was 10.2 years (range 0.1–19.4 years). The tumours were graded according to Fuhrman. Representative histological sections were stained for CD31, which decorates endothelial cells, in order to assess microvascular invasion (MVI). In univariate analysis, microvascular invasion (p<0.01), tumour size (TS) (p=0.01), TNM stage (p=0.01) and Fuhrman nuclear grade (p=0.02) were significant predictors of cancer‐specific survival. Multivariate analysis, adjusted for age, revealed that microvascular invasion, tumour size and nuclear grade were independent covariates. According to our findings microvascular invasion is a strong independent prognostic predictor, and including this in the histopathology report should be considered together with nuclear grade and tumour size.

LI, ZHENFENG; LIU, JINZHONG; TANG, FUAI; LIU, YIQING; WALDUM, HELGE L.; CUI, GUANGLIN

doi: 10.1111/j.1600-0463.2008.01048.xpmid: 19133005

Histamine is produced by mast cells and many other types of cells. The role of histamine released from mast cells in promoting tumor angiogenesis has been intensively studied; however, the role of non‐mast cell histamine in regulating tumor angiogenesis has been largely ignored. In this study, tissue specimen sections from 43 patients with esophageal squamous cell carcinoma (ESCC) and normal esophageal biopsies from 17 heath individuals obtained from a high incidence area of north China were used to assess changes in microvessel density (MVD) and non‐mast cell L‐histidine decarboxylase (HDC) (the only rate‐limiting enzyme that catalyzes the formation of histamine from L‐histidine) expression in the tumor microenvironment by immunohistochemistry (IHC). In addition, the cellular characterization of non‐mast cell HDC‐positive cells in microvessels was examined by double IHC combined with HDC/CD34 and HDC/PCNA antibodies. These IHC analyses revealed a significantly increased HDC‐positive MVD in ESCC as compared with normal controls, which accounted for ∼61% of CD34‐labeled general MVD in ESCC. Furthermore, IHC in serial sections and double IHC showed that most of these HDC‐positive cells were CD34‐positive endothelial cells in microvessels with an increased proliferative capacity. Thus, our results suggest that non‐mast cell histamine expressed in endothelial cells of microvessels could be an additional cellular source and might play a role in regulating angiogenesis in ESCC.

KING, PAUL; NGUI, JAMES; OPPEDISANO, FRANCES; ROBINS‐BROWNE, ROY; HOLMES, PETER; HOLDSWORTH, STEPHEN

doi: 10.1111/j.1600-0463.2008.01078.xpmid: 19133006

Nontypeable Haemophilus influenzae (NTHi) is a mucosal pathogen that is a major cause of respiratory infection, including sinusitis, otitis media and bronchitis. This bacterium has evolved a number of mechanisms to facilitate its survival in the human host. Recently it has been recognized that it is capable of intracellular survival in monocytes/macrophages and epithelial cells. Previous work by the authors has demonstrated that the protective response to NTHi is Th1 predominant. This information led to the hypothesis that the intracellular survival of NTHi in human monocytes may be reduced by two key effector mechanisms of Th1‐mediated immunity: interferon gamma and ligation of CD40. This study assessed the effect of interferon gamma and ligation of CD40 on the intracellular survival of NTHi in human monocytes. Responses were studied in monocytes from subjects with bronchiectasis and persistent airway infection with NTHi and compared with control subjects. The results demonstrated that different isolates of NTHi were able to survive inside monocytes. Killing of one strain of NTHi could be enhanced by the addition of interferon gamma and CD40 ligation in both control and bronchiectasis subjects. Other strains were more resistant.

BRIEGER, JUERGEN; DUESTERHOEFT, ANNE; BROCHHAUSEN, CHRISTOPH; GOSEPATH, JAN; KIRKPATRICK, C. JAMES; MANN, WOLF J.

doi: 10.1111/j.1600-0463.2008.01088.xpmid: 19133007

The predictive value of cadherin‐11, tenascin, fascin, and mucin‐1 as markers for the likelihood of recurrence in pleomorphic adenoma of the parotid gland was examined. In this retrospective study we analysed 20 tumours from16 patients by immunohistochemistry. Staining intensities were measured using a semiquantitative scoring approach; localisation (tumour centre vs border) as well as clinical data were analysed and correlated with follow‐up. Cadherin‐11 was increased in recurrent tumours. However, no changes of fascin, tenascin or mucin‐1 were observed. Cadherin‐11 and fascin were increased in primary tumours of patients with later recurrence, with fascin upregulation restricted to the tumour border. In conclusion, cadherin‐11 and fascin should be further analysed for their value as markers for later recurrence in pleomorphic adenoma. Our observations might reflect dysregulation of cellular pathways contributing to cellular dissemination, which might potentially result in later recurrence.

SMOLEJ, L.; KAŠPAROVÁ, P.

doi: 10.1111/j.1600-0463.2008.00987.xpmid: 19133008

Angiogenesis is a potential prognostic factor in chronic lymphocytic leukemia (CLL). Elevated circulating levels of angiogenic factors in CLL have been repeatedly reported. Nevertheless, the issue of bone marrow neovascularization in CLL remains controversial, partly due to limited number of published studies, different methods of assessing microvessel density (MVD) and small patient cohorts. Moreover, there are very scarce data regarding the relationship of marrow angiogenesis to prognostic markers in CLL. Our objectives were: 1. To assess bone marrow MVD in CLL using two different monoclonal antibodies and a reproducible method of MVD quantification; 2. To examine the possible association of marrow MVD and clinical course, pattern of marrow infiltration, Rai stage, cytogenetic abnormalities detected by fluorescence in situ hybridization (FISH), and mutation status of immunoglobulin heavy chain variable region (IgVH). MVD was higher using CD34 vs vWF antibody (p<0.0001). However, no MVD differences were detected between CLL subgroups subdivided according to the above‐mentioned prognostic factors. In conclusion, MVD assessment using anti‐CD34 resulted in higher MVD counts than when using anti‐vWF antibody. No association of MVD with any prognostic factors was observed, possibly due to the limited patient cohort. As the need for bone marrow trephine biopsies in CLL is significantly decreasing, a standardized method of neovascularization assessment is required to enable possible multicentre studies in order to conduct larger investigations and thereby shed more light on the real clinical significance of bone marrow angiogenesis in CLL.

GURSOY, ULVI KAHRAMAN; KÖNÖNEN, EIJA; UITTO, VELI‐JUKKA

doi: 10.1111/j.1600-0463.2008.00868.xpmid: 19133009

We examined survival and replication of fusobacteria inside epithelial cells. Subconfluent cultures of HaCaT keratinocytes were infected with five bacterial strains representing three Fusobacterium species: F. nucleatum, F. necrophorum, and F. mortiferum. Adhesion and invasion of the bacteria were assayed before and after antibiotic treatment that killed the adhered and extracellular bacteria. The number of live fusobacteria was examined by bacterial culturing after sonication of the epithelial cells. The role of host cell cytoskeleton functions was examined by treating the epithelial cells with cell function inhibitors. Number of viable epithelial cells was measured with the CellTiter96 kit. The tested Fusobacterium species adhered to and invaded the epithelial cells, and multiplied intracellularly for several hours. Thereafter, the intracellular number of bacteria rapidly declined. Concomitantly, viable fusobacteria were detected in the culture medium. Treatment of the infected epithelial cells with an actin formation inhibitor markedly reduced the number of living intracellular fusobacteria. Newly formed actin filaments were seen by confocal microscopy in the epithelial cells associated with the invaded bacteria. Fusobacteria infection did not reduce the number of viable epithelial cells in culture. Thus, fusobacteria are able to adhere to and invade epithelial cells, and survive under aerobic conditions. This property may enable them to survive in mucosa and participate in various disease processes of oral and pharyngeal tissues.

WANG, LI‐MEI; BAI, YIN‐LAN; SHI, CHANG‐HONG; GAO, HUI; XUE, YING; JIANG, HONG; XU, ZHI‐KAI

doi: 10.1111/j.1600-0463.2008.01095.xpmid: 19133010

Developing a new generation of vaccines is important for preventing tuberculosis (TB). DNA vaccine is one promising candidate. In this study we evaluated the immunogenicity and protective efficacy of the DNA vaccine encoding the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) with human interleukin‐2 (hIL‐2) in BALB/c mice. We showed that the DNA vaccine pcDNA‐Hsp65‐hIL‐2 could induce high levels of antigen‐specific antibody, IFN‐γ, CD4+ and CD8+ T cell production. When the immunized mice were infected with M. tuberculosis H37Rv, the organ bacterial loads in the DNA immunized group were significantly reduced compared to those of the saline control group, but the ability to reduce bacteria was not better than for BCG. The histopathology in lungs of the DNA vaccine immunized mice was similar to that of BCG immunized mice, which was obviously ameliorated compared to that of the saline control group. Overall, the DNA vaccine could afford protection against M. tuberculosis infection, though the protection efficacy was not as great as that of conventional BCG.

HÖGDAHL, M.; SÖDERLUND, G.; KIHLSTRÖM, E.

doi: 10.1111/j.1600-0463.2008.01145.xpmid: 19133011

Chlamydia pneumoniae has during recent years been associated with cardiovascular disease and atherosclerosis. Chemokines, leukocyte adhesion proteins and metalloproteinases are significant for chemotaxis and attachment of leukocytes to vessel walls, and for stability of atherosclerotic plaques. To determine the ability of C. pneumoniae to elicit inflammation in a relevant target host cell, we infected human coronary artery endothelial cells (HCAEC) with a clinical isolate of C. pneumoniae. Extracellular release of five chemokines, two adhesion proteins and a metalloproteinase was measured at different time points after infection using a cytometric bead assay and ELISA. Secretion of IL‐8, MCP‐1, MIG, IP‐10 and ICAM‐1 was significantly increased 48 h after C. pneumoniae infection of HCAEC in comparison with uninfected controls. Release of RANTES occurred already 6 h after infection. C. pneumoniae did not elicit release of E‐selectin or MMP‐1. We conclude that C. pneumoniae induces expression of proinflammatory components in HCAEC, which would promote migration of leukocytes towards endothelial cells. This suggests that C. pneumoniae initiates and propagates vascular inflammation in ways that contribute to coronary artery disease.

ZHANG, YE; LI, XIN‐HONG; JIANG, HONG; HUANG, CHANG‐XING; WANG, PING‐ZHONG; MOU, DAN‐LEI; SUN, LI; XU, ZHE; WEI, XIN; BAI, XUE‐FAN

doi: 10.1111/j.1600-0463.2008.01011.xpmid: 19133012

Hantaan virus (HTNV) is an Old World hantavirus associated with hemorrhagic fever with renal syndrome (HFRS). To visualize the localization of the L protein of HTNV strain 84FLi within cells, a fusion protein composed of enhanced green fluorescent protein and L protein, EGFP‐L, was expressed in Vero cells. The 273 KDa expressed fusion protein of EGFP‐L localized in the perinuclear region. We also described the development of a reverse genetics system for HTNV strain 84FLi. The RNA polymerase I (pol I)‐mediated transcription system was used to generate artificial viral RNA genome segments (minigenomes), which contained the chloramphenicol acetyltransferase (CAT) reporter gene in antisense (virus RNA) or sense (virus‐complementary RNA) orientation flanked by the noncoding regions of HTNV 84FLi L segment. CAT could be detected in cells after transfection, indicating the successful encapsidation, transcription and replication of the pol I‐derived minigenomes. The passaged transfer of CAT demonstrates that recombinant virus containing packaged pol I‐derived minigenomes has been produced. This system may be helpful in studying the gene function and pathogenesis of HTNV.

MIYAWAKI, MICHIYO; HIJIYA, NAOKI; TSUKAMOTO, YOSHIYUKI; NAKADA, CHISATO; KAWAHARA, KATSUNOBU; MORIYAMA, MASATSUGU

doi: 10.1111/j.1600-0463.2008.01125.xpmid: 19133013

This study aimed to determine whether epidermal growth factor receptor (EGFR), which has been reported to be frequently overexpressed in esophageal carcinoma cells, is actually activated in the cells. Paraffin‐embedded specimens of 39 cases of esophageal carcinoma were analyzed immunohistochemically with anti‐EGFR polyclonal antibody (α‐EGFR Ab) and also an anti‐phospho‐EGFR‐specific polyclonal antibody (α‐p‐EGFRTyr992 Ab) that specifically recognizes phosphorylated tyrosine 992 of EGFR. All of the 39 cases were found to express EGFR, but the expression levels were not significantly higher than those in basal cells of the normal esophageal epithelium. In 38 of the 39 cases, α‐p‐EGFRTyr992 immunoreactivity was evident. Interestingly, the positively stained carcinoma cells were not distributed diffusely, and strongly immunostained cells tended to be localized in areas of severe dysplasia and in microinvasive foci just adjacent to the main invasive carcinoma. However, the deeply invasive front never exhibited positive immunoreactivity. The present findings suggest that phosphorylation of EGFR Tyr992 may play some specific functional role in esophageal carcinomas besides promotion of cell proliferation.