TP53 regulates human AlkB homologue 2 expression in glioma resistance to Photofrin-mediated photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) is a promising adjuvant therapy in cancer treatment. However, cancers resistant to PDT, mediated through the efflux of photosensitisers by means of P-glycoprotein or ATP-binding cassette transporter proteins, have been reported. The DNA repair has also been suggested to be responsible for PDT resistance, but little is known about the repair pathways and mechanisms involved. Therefore, this study aimed to investigate the possible function of six major DNA repair mechanisms in glioma cells resistant to Photofrin-mediated PDT (Ph-PDT). METHODS: The U87 glioma cells relatively resistant to Ph-PDT were obtained by recovering the viable cells 3 h after PDT treatment. The mRNA and protein expression levels of DNA repair genes were evaluated by quantitative real-time reverse transcription-polymerase chain reaction and western blotting, respectively. Small-interfering RNA and chromatin-immunoprecipitation assays were used to further examine the relationship between AlkB, an alkylation repair homologue 2 (Escherichia coli) (ALKBH2) and Ph-PDT responsiveness, and transcription factors involved in ALKBH2 transcription. RESULTS: The ALKBH2 of DNA damage reversal was significantly increased at both mRNA and protein levels from 30 min to 48 h post-treatment with Ph-PDT. Conversely, down-regulating ALKBH2 expression enhances Ph-PDT efficiency. Furthermore, our data clearly show for the first time that tumour protein (TP53) is directly involved by binding to the promoter of ALKBH2 in mediating Ph-PDT resistance. CONCLUSION: C The DNA damage reversal mechanisms may have important functions in Ph-PDT resistance through the activation of ALKBH2 by TP53.

Severe acute respiratory syndrome outbreak promotes a strong sense of professional identity among nursing students.

This is a phenomenological study that examined nursing students' perception of nursing professional identity during severe acute respiratory syndrome (SARS) outbreak in Hong Kong in 2003. The aim of the study was to find out how the impact of the SARS event might have affected nursing students in identification with the nursing profession. A total of 10 nursing students were interviewed. This study showed that the SARS crisis enhanced a reconstruction of worldview and affirmed the professional identity of nursing students. Central themes derived from the interview were (1) appreciation and sharing of nursing identity; (2) a sense of moral duty; (3) a change of worldview and feeling of self-growth. This study provided insights to nursing education that acquisition of professional identity could be enhanced through reflective appreciation of critical events such as SARS.

Antisense epidermal growth factor receptor RNA transfection in human glioblastoma cells down-regulates telomerase activity and telomere length.

Epidermal growth factor receptor is overexpressed and/or amplified in up to 50% of glioblastomas, suggesting an important role of this gene in glial tumorigenesis and progression. In the present study we demonstrated that epidermal growth factor receptor is involved in regulation of telomerase activity in glioblastoma. Antisense-epidermal growth factor receptor approach was used to inhibit epidermal growth factor receptor expression of glioblastoma U87MG cells. Telomerase activity in antisense-epidermal growth factor receptor cells decreased by up to 54 folds compared with control cells. Moreover, the telomere lengths of antisense-epidermal growth factor receptor cells were shortened. In addition, the tumorigenicity of antisense-epidermal growth factor receptor cells was significantly inhibited. Taken together, there were strong correlations between tumorigenicity and epidermal growth factor receptor expression levels, and between tumorigenicity and telomerase activity. These results provide evidence that epidermal growth factor receptor plays an important role in the regulation of telomerase activity of glioma cells. Our findings provide new insights into both the biological functions of epidermal growth factor receptor and the regulation of telomerase activity. The inhibition of telomerase activity triggered by antisense-epidermal growth factor receptor treatment may reflect yet another mechanism of antisense-epidermal growth factor receptor approach in tumour suppression.

93G, a novel sporadic strain of hepatitis E virus in South China isolated by cell culture.

The nucleotide sequence of Hepatitis E virus (HEV) serous isolates (G-9 and G-20) from Guangzhou, South China, which has been reported previously, are divergent significantly from those of other reported HEV isolates. In order to investigate more extensively the Guangzhou isolate, the 93G strain was isolated from the faecal sample of the same individual as G-9 by A549 cell culture and identified immunologically and by molecular biological techniques. The results showed that strain 93G could be propagated in an A549 cell line causing cytopathic effects. The viral particles were aggregated by a specific antibody to HEV Chinese Xinjiang strain (87A) observed using immunoelectron microscopy and were similar morphologically to HEV from other sources. In this study, an indirect fluorescent antibody assay was first developed to examine HEV antigen in the infected cells, by immunofluorescence in the cytoplasm and on the surface membrane of the cells. The 58-kDa and 82-kDa native structural proteins of HEV were also identified in this study by Western blotting. The 93G genome showed high homology (93%) with G-9 previously reported but was also as divergent from the Burmese, Mexican, Chinese Xinjiang isolates and the recently reported US-1 isolate, as was G-9. The data presented indicate that 93G propagated in A549 cells, together with its related serum isolate G-9, represents another HEV strain circulating in China and is responsible for some sporadic hepatitis E infections.

Restoration of wild-type PTEN expression leads to apoptosis, induces differentiation, and reduces telomerase activity in human glioma cells.

PTEN is a candidate tumor suppressor gene identified on human chromosome 10q23.3 that is frequently mutated or deleted in 30% to 44% of glioblastomas. Transient expression study of PTEN in glioma cells indicates that PTEN plays an important role in cellular proliferation, tumorigenicity, cell migration, and focal adhesions. In this study, we examined the biological consequences on U87MG glioma cells after stable gene transfer of wild-type PTEN. Cells stably expressing wild-type PTEN protein were found to have suppressed proliferation, as determined by cell counting and Ki-67 staining, as well as inhibited anchorage-independent growth. The PTEN-expressing cells also showed higher expression of glial fibrillary acidic protein and changed morphologically from spindle-shaped to elongated cell bodies with multiple slender processes, suggesting that these cells have undergone differentiation. In addition, telomerase activity decreased more than 10-fold in PTEN-expressing cells when compared with control cells. More importantly, apoptosis was detected in about 5% of PTEN-expressing cells, representing a 17-fold (p < 0.01) increase over the control cells. Taken together, these results suggest that PTEN plays an important role in regulation of cell homeostasis by maintaining a balance between proliferation, differentiation, and apoptosis.

Antisense epidermal growth factor receptor RNA transfection in human malignant glioma cells leads to inhibition of proliferation and induction of differentiation.

The epidermal growth factor receptor (EGFR) is a protooncogene that is frequently observed with alterations in late stage gliomas, suggesting an important role of this gene in glial tumorigenesis and progression. In this study we evaluated an antisense EGFR approach as an alternative therapeutic modality for glioblastomas. We transfected U-87MG cells with an antisense EGFR construct and obtained several clones stably expressing lower or undetectable levels of EGFR protein. These clones were found to have impaired proliferation as well as a reduced transforming potential to grow in soft agarose. The number of cells positive for the cell cycle-specific nuclear antigen Ki-67 was also significantly decreased (P < 0.05) in antisense EGFR-transfected clones compared with parental or empty vector-transfected cells. Flow cytometric analysis revealed that the proportion of cells in G0/G1 phases of the cell cycle in the antisense clones increased by up to 31% compared with control cells, whereas the proportion of cells in S phase decreased by up to 58%. In addition, the antisense EGFR-transfected cells showed higher expression of glial fibrillary acidic protein and a more differentiated form, with smaller cell bodies possessing fine tapering cell processes. These results suggest that EGFR plays a major role in modulating cell growth and differentiation in glioblastoma cells. Our experimental model of antisense EGFR provides a basis for future development of antisense EGFR oligodeoxynucleotides in treatment of glioblastomas.

Early E-selectin, VCAM-1, ICAM-1, and late major histocompatibility complex antigen induction on human endothelial cells by flavivirus and comodulation of adhesion molecule expression by immune cytokines.

Expression of E-selectin (ELAM-1, CD62E) on human umbilical vein endothelial cells significantly increased 30 min postinfection with the flavivirus West Nile virus (WNV), was maximal by 2 h postinfection, and declined to baseline levels within 24 h. Expression of ICAM-1 (CD54) and VCAM-1 (CD106) was significantly increased by 2 h and maximal at 4 h after infection. P-selectin (CD62P) expression was unaffected by WNV. Upregulation occurred earlier than that caused by tumor necrosis factor alpha (TNF-alpha) or interleukin 1 (IL-1) and could not be inhibited by neutralizing TNF-alpha, IL-1alpha, or alpha/beta interferon (IFN-alpha/beta) antibodies, suggesting a direct, virus-mediated phenomenon. TNF-alpha significantly enhanced WNV-induced increases in E-selectin, P-selectin, ICAM-1, and VCAM-1 expression, while IFN-gamma enhanced WNV-induced ICAM-1 expression. In contrast, IL-4 abrogated WNV-induced E-selectin expression increases but acted in synergy with WNV to increase P-selectin and VCAM-1 expression. WNV increased the expression of class I and II major histocompatibility complex antigens (MHC-I and MHC-II, respectively) at 24 and 72 h, respectively. IFN-gamma, TNF-alpha, or IL-1 acted in synergy with WNV to produce greater increases in MHC-I expression than WNV or cytokines alone, while IFN-alpha/beta or IL-4 had no effect. MHC-II induction in cytokine-treated, WNV-infected cells was similar to that caused by cytokines alone. Neutralizing IFN-alpha/beta antibody inhibited WNV-induced MHC-I expression by 30% at 24 h and by 100% by 72 h. The differential kinetics of modulation suggest sequential adhesion of leukocyte subpopulations to infected endothelial cells, which may be important in initial viral spread in vivo.

Regulation of adhesion molecule expression by human synovial microvascular endothelial cells in vitro.

OBJECTIVE: To examine the in vitro expression of E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1), ICAM-2, vascular cell adhesion molecule 1 (VCAM-1), and platelet-endothelial cell adhesion molecule 1 (PECAM-1) by synovial microvascular endothelial cells (SMEC) in comparison with microvascular neonatal foreskin endothelial cells (FSE) and macrovas- cular human umbilical vein endothelial cells (HUVE). METHODS: Cultured endothelial cells were treated for 4 hours with medium alone or tumor necrosis factor alpha (TNF alpha). The expression of endothelial adhesion molecules was evaluated by flow cytometry, cell enzyme-linked immunosorbent assay, and Northern blot analysis. RESULTS: SMEC continuously expressed E-selectin under basal culture conditions, whereas FSE and HUVE did not. TNF alpha treatment of rheumatoid arthritis (RA) SMEC resulted in sustained peak expression of E- selectin for up to 24 hours, which subsequently declined but remained elevated even at 72 hours. In contrast, peak E-selectin expression in FSE and HUVE occurred between 4 hours and 16 hours after TNF alpha treatment and then declined to near basal levels by 24-48 hours. SMEC expressed significantly higher levels of ICAM-1 compared with HUVE under basal culture conditions. There was no difference between SMEC, FSE, and HUVE in the expression of P-selectin, VCAM-1, ICAM-2, or PECAM-1. Northern blot analysis demonstrated that the levels of E-selectin expression by TNF alpha stimulated endothelial cells correlated with their respective messenger RNA levels. CONCLUSION: Regulation of E-selectin and ICAM-1 expression in RA synovial endothelium is different from that in neonatal foreskin and human umbilical vein endothelium. The augmented expression of adhesion molecules in RA synovial endothelium may facilitate the recruitment of leukocytes to this site.

Effect of gold sodium thiomalate and its thiomalate component on the in vitro expression of endothelial cell adhesion molecules.

Endothelial adhesion molecules play an important role in the tissue recruitment of leukocytes in inflammatory conditions such as rheumatoid arthritis. We have investigated the effect of the antirheumatic drug gold sodium thiomalate on adhesion molecule protein and mRNA expression in cultured human endothelial cells. Gold sodium thiomalate inhibited cytokine (TNF, IL-1, IL-4)-stimulated expression of vascular cell adhesion molecule-1 and E-selectin but not intercellular adhesion molecule-1 on endothelial cells. Gold sodium thiomalate also suppressed TNF-stimulated increases in vascular cell adhesion molecule-1 and E-selectin mRNA levels but had no effect on intercellular adhesion molecule-1 mRNA. Thiomalate (mercaptosuccinate), but not gold thioglucose or D-penicillamine, mimics the effect of gold sodium thiomalate at equimolar concentrations. We propose that the inhibition of vascular cell adhesion molecule-1 and E-selectin expression by gold sodium thiomalate is due to its thiomalate and not its gold component. Gold sodium thiomalate has a direct effect on endothelial adhesion molecule expression, and this may contribute to its antiinflammatory activity.

Augmented lymphocyte binding to cultured endothelium in psoriasis.

Lymphocyte binding to cultured human umbilical vein endothelial cells was evaluated using a modified centrifugation binding assay in 15 patients with psoriasis and compared with three patients with atopic dermatitis, 11 patients with rheumatoid arthritis and 28 normal controls. Patients with psoriasis demonstrated 61% augmented lymphocyte binding compared with normal controls (P < 0.0001), which was not explained by differences in age and sex or an effect of psoriatic sera. In serial studies of six patients, this difference was found to be reversible with treatment and clinical improvement. Lymphocytes from patients with atopic dermatitis demonstrated decreased binding to endothelium (P < 0.005), while those from patients with rheumatoid arthritis were not different from normal controls. This is the first skin disease described in which augmented lymphocyte binding to endothelium occurs, and may represent a mechanism by which lymphocytes are targeted to psoriatic skin.

Identification of a human endothelial cell activation antigen that is co-expressed by germinal follicle centre B lymphocytes.

Endothelial cell activation antigens may play important roles in immune responses and in inflammation. This report describes the identification and characterization of a monoclonal antibody, named EAA-B, which reacts specifically with human umbilical vein endothelial (HUVE) cells pre-treated with tumour necrosis factor-alpha (TNF-alpha) but not with untreated cells. The expression of the EAA-B antigen on HUVE cells could also be induced by interleukin-1 (IL-1), bacterial lipopolysaccharide (LPS), and phorbol esters but not by interferon-gamma (IFN-gamma). By contrast, EAA-B antigen expression on neonatal foreskin and rheumatoid synovial fibroblasts, whether pre-treated with TNF-alpha or not, was not detectable. Peripheral blood leucocytes and the leukaemic cell lines U937, HL-60, Raji and Molt 4 showed no detectable expression of the EAA-B antigen. Kinetic studies demonstrated that the EAA-B antigen was rapidly expressed, peaked at 6 hr and declined to basal level by 24 hr. Western blotting revealed that monoclonal antibody EAA-B recognized a polypeptide of approximately 80,000-90,000 MW. EAA-B partially blocked the augmented adhesion of HL-60 cells to TNF-treated HUVE cells. However, it failed to inhibit the enhanced binding of peripheral blood leucocytes, U937, Raji and Molt 4 Cells to TNF-treated HUVE cells. In situ, the EAA-B antigen was detected on some vascular endothelium in tonsils, lymph nodes, psoriatic skin and rheumatoid synovium but not in normal non-lymphoid tissues. Interestingly EAA-B antigen is also expressed by B lymphocytes in germinal follicle centres (GFC) of lymphoid tissues. The co-expression of this endothelial activation antigen by GFC B lymphocytes may have significant implications for immune responses and in B-lymphocyte differentiation.

An animal investigation into the effects of tympanostomy on middle ear mucociliary function and cilia.

The effect of tympanostomy on guinea-pig middle ear mucosa with particular reference to mucociliary function and cilia was investigated. 13 guinea-pigs were used; 5 acted as the control group with no surgical intervention whilst the other 8 had perforations made in one tympanic membrane. 6 weeks later an attempt was made to measure mucociliary function in all the ears, but this was unsuccessful. However, it would appear that fashioning a perforation in the tympanic membrane causes no histological changes in this animal model.

A 'silent' intracranial complication of frontal sinusitis.

Intracranial complications of frontal sinusitis, although rare today, do still develop despite widespread use of antibiotics. We report a case which demonstrates how silently a frontal lobe abscess may present with subtle changes in mood and behaviour, with no focal neurological signs. Diagnosis and management are discussed and a brief review of the incidence of intracranial complications of frontal sinusitis, mode of spread, clinical presentation, investigations, treatment and bacteriology is presented.

Effect of leukotriene B4 and prostaglandin E2 on the adhesion of lymphocytes to endothelial cells.

The arachidonic acid metabolites leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) may play an important role in inflammation. It is not known whether these mediators influence the binding of lymphocytes to endothelial cells, a process which is important in the extravasation of lymphocytes in inflammatory states. In the present investigation, the effect of LTB4 and PGE2 on the binding interaction between lymphocytes and endothelial cells was examined using a centrifugation cell binding assay. Although LTB4 elicited an aggregation response on human polymorphonuclear leucocytes (PMNL) and enhanced their binding to endothelial cells it had no effect on lymphocyte binding. By contrast, PGE2 caused a dose-dependent inhibition of lymphocyte binding to endothelial cells. The inhibitory effect of PGE2 had a rapid onset but was exhibited only when PGE2 was present continuously during the cell binding assay. Although the mechanism by which PGE2 acts is not clear, it may provide a negative feedback mechanism in regulating the influx of lymphocytes into inflammatory sites.

Hypothesis: lymphocyte trafficking in inflammatory rheumatic disease. A role for receptor mediated homing.

The entry of lymphocytes into normal lymph nodes occurs principally after their passage across postcapillary high endothelial venules. Nonlymphoidal tissues do not normally express high endothelial venules; however, in inflammatory rheumatic diseases associated with prominent lymphocyte accumulation (e.g., rheumatoid synovium), high endothelial venule-like structures are found and may facilitate local lymphocyte traffic. In mouse and man there are lymphocyte surface membrane homing receptors which determine lymphocyte binding to acceptor sites on lymph node high endothelial venules. The migration of lymphocytes from the circulation into inflamed nonlymphoidal tissues may be controlled by similar homing receptor mediated mechanisms.

Traumatic neuroma of the internal laryngeal nerve as a cause of severe cough.

We present a case of traumatic neuroma, describing an unusual symptom which may be more common than is recognized, and indeed we think from our search of the literature that this is the first report of such a case. We have briefly reviewed the pathology, clinical presentation and treatment of traumatic neuromas.

A prospective trial of unilateral grommets for bilateral secretory otitis media in children.

Fifty-four children with established chronic secretory otitis media, who had failed to respond to medical measures were treated with adenoidectomy and insertion of 1 grommet on a side chosen at random. Both sides improved and remained significantly improved at 12 months (P less than 0.001). At 3 months, the side with the grommet improved significantly more than the other side (P less than 0.05) but at 12 months there was no significant difference between the 2 sides (P greater than 0.1).