Cytopathic effects of Treponema denticola chymotrypsin-like proteinase on migrating and stratified epithelial cells.

The effects of Treponema denticola and its outer membrane-bound chymotrypsin-like proteinase on periodontal ligament epithelial cell cultures at different stages of maturity were studied. In sparse cultures with migrating epithelial cells, large intracellular vacuoles were formed rapidly following exposure to live T. denticola. Treponemes showing structural damage were seen occasionally inside membrane-bound vesicles. Intensive membrane blebbing occurred in infected cells and continued for up to 48 h before the cell died. Blebbing could also be induced by a purified chymotrypsin-like proteinase of T. denticola. Cortical actin and alpha-actinin of the bacterium-treated cells showed disorganization, and pericellular fibronectin was degraded by both whole T. denticola and the isolated proteinase. Epithelial cells with well-formed lateral cell contacts appeared to be more resistant to the effects of T. denticola than migrating isolated cells. In multilayer epithelial cultures, adhesion of T. denticola and membrane blebbing were observed infrequently. There was no evidence of invasion of T. denticola into epithelial multilayers. However, immunogold electron microscopy showed rapid transport of T. denticola chymotrypsin-like proteinase into newly formed large intracellular vacuoles within the epithelial layers. These vacuoles were lined by membranes studded with ribosomes. T. denticola-treated epithelial multilayers had loose cell contacts, collapsed intercellular spaces, and increased permeability. Through its capacity to cause these unique cytopathic effects, the chymotrypsin-like proteinase of T. denticola has the potential to contribute to the initiation of periodontal disease.     

Immunohistochemical and ultrastructural correlates of muscle-actin expression in pleomorphic adenomas and myoepitheliomas based on comparison of formalin and methanol fixation

Summary The degree and range of differentiation of the cells referred to as myoepithelial-like in pleomorphic adenomas and the tumour cells of myoepitheliomas are not definitely established. This type of information is critical for establishing reliable diagnostic criteria, such as expression of muscle-specific actin and ultrastructural identification of myofilaments, in these and other salivary gland tumours. Pleomorphic adenomas (18) and myoepitheliomas (5), of which 10 cases were fixed only in formalin and 13 cases where tissues were fixed in both formalin and methanol/acetic acid, were studied. Each tumour and normal accompanying parotid was immunostained with two monoclonal antibodies for smooth muscle actin, HHF35 and MSA. Staining of myoepithelial cells was absent in certain samples of normal gland with both HHF35 (15%) and MSA (69%) when formalin-fixed tissue was used. Using formalin-fixed tissue from 15 pleomorphic adenomas/myoepitheliomas, 2 (14%) had focal positivity with HHF35, while 8 cases (57%) were positive with MSA. However, a certain degree of false positivity was suspected since in samples of normal parotid, both acinar and duct cells were frequently stained, particularly with MSA. With methanol/ acetic acid-fixed tissue only 4 of 13 cases (31%) were positive with either MSA or HHF35 and 2 of these only had a minor proportion of the tumour cells expressing muscle-specific actin. Using alcohol-fixed tissue, myoepithelial cells were strongly stained in all examples of normal parotid gland with both anti-actin antibodies. In 5 cases examined by electron microscopy, there was no apparent correlation between immunohistochemical results and the presence or absence of cytoplasmic filament accumulation. The results indicate considerable tumour cell heterogeneity in muscle-specific actin expression and suggest that non-luminal cells in pleomorphic adenomas and the tumour cells in myoepitheliomas may differentiate as classical myoepithelial cells, as partially differentiated (i.e. modified myoepithelial cells) or as the counterpart of basal cells present in the intra- and interlobular ducts of normal salivary gland.     

Systemic oxidative and antioxidative status in Chinese patients with asthma

BACKGROUND: Patients with asthma generate an increased amount of reactive oxygen species from peripheral blood cells. Reactive oxygen species produce many of the pathophysiologic changes associated with asthma and may contribute to its pathogenesis. OBJECTIVE: We investigated changes in antioxidant enzyme activities and oxidized glutathione (glutathione disulfide; GSSG) levels in erythrocytes from a group of healthy control Chinese subjects (n=135) and patients with asthma (n=106). METHODS: Baseline pulmonary function was measured for all subjects. Antioxidant status was evaluated by measuring erythrocyte superoxide dismutase, catalase, and glutathione peroxidase activities. Oxidative stress was also measured in terms of GSSG in erythrocytes with a kinetic microassay. RESULTS: Patients with asthma had significantly increased erythrocyte superoxide dismutase and catalase activities compared with controls (61.10 +/- 1.30 U/g hemoglobin [Hb] vs 55.51 +/- 1.82 U/g Hb [P=.018] and 0.0637 +/- 0.0021 U/g Hb vs 0.0257 +/- 0.0120 U/g Hb [P <.001] for the asthma and control groups, respectively). Conversely, erythrocyte glutathione peroxidase activity decreased (44.21 +/- 1.33 mU/g Hb vs 50.07 +/- 1.39 mU/g Hb for the asthma and control groups, respectively; P=.003). Patients with asthma also had significantly higher GSSG levels in erythrocyte hemolysates compared with controls (167.40 +/- 2.93 micromol/L vs 44.98 +/- 0.44 micromol/L for the asthma and control groups, respectively; P <.001), indicating increased oxidative stress. CONCLUSIONS: Asthma is accompanied by an alteration in systemic antioxidant status due to possible oxidative stress in this disease.     

Synergistic effect of IL-4 and TNF-alpha in the induction of monocytic differentiation of a mouse myeloid leukaemic cell line (WEHI-3B JCS).

We have previously shown that non-cytotoxic concentrations (600-1200 U/ml) of recombinant mouse tumour necrosis factor-alpha (TNF-alpha) can induce differentiation of a subclone (JCS) of the WEHI-3B myelomonocytic leukaemia cell line into mature cells with the characteristics of macrophages. In the present study, the effects of recombinant mouse interleukin-4 (IL-4), either alone or in combination with mouse TNF-alpha, on the growth and differentiation of JCS cells were examined. IL-4 alone (20-5000 U/ml) inhibited the growth of JCS cells in a dose-dependent manner but did not induce cell differentiation. However, combinations of IL-4 and TNF-alpha acted in synergy to inhibit cell proliferation and induce monocytic differentiation of JCS cells, as shown by increased expression of the macrophage differentiation antigens (F4/80, Mac-1), stimulation of phagocytic activity, induction of non-specific esterase and NBT-reducing activities, increased plastic adherence and morphological criteria. Similar synergistic interactions were also shown by human TNF-alpha and mouse IL-4, indicating that TNF-alpha might exert its effects through the low-affinity (p55) TNF receptors. Moreover, the clonogenicity of JCS cells in vitro and their tumorigenicity in vivo were significantly reduced by combined TNF-alpha and IL-4 treatment. Our results indicate that TNF-alpha can act as a differential signal for JCS cells and that its effects are modulated by IL-4. Therefore, the combination of TNF-alpha and IL-4 may be useful in the treatment of some forms of myelomonocytic leukaemia.     

The major surface protein complex of Treponema denticola depolarizes and induces ion channels in HeLa cell membranes.

The oral spirochete Treponema denticola is closely associated with periodontal diseases in humans. The 53-kDa major surface protein (Msp) located in the outer membrane of T. denticola serovar a (ATCC 35405) has both pore-forming activity and adhesin activity. We have used standard patch clamp recording methods to study the effects of a partially purified outer membrane complex containing Msp on HeLa cells. The Msp complex was free of the chymotrypsin-like proteinase also found in the outer membrane of T. denticola. Msp bound to several HeLa cell proteins, including a 65-kDa surface protein and a 96-kDa cytoplasmic protein. The Msp complex depolarized and increased the conductance of the HeLa cell membrane in a manner which was not strongly selective for Na+, K+, Ca2+, and Cl- ions. Cell-attached patches of HeLa cell membrane exposed to Msp complex exhibited short-lived channels with a slope conductance of 0.4 nS in physiologically normal saline. These studies show that Msp binds both a putative epithelial cell surface receptor and cytoplasmic proteins and that the Msp complex can form large conductance ion channels in the cytoplasmic membrane of epithelial cells. These properties may contribute to the cytopathic effects of T. denticola on host epithelial cells.     

Comparison of three different sensitive assays for hepatitis B virus DNA in monitoring of responses to antiviral therapy

The aim of our study was to compare the performances of two new hepatitis B virus (HBV) DNA assays, a cross-linking assay (NAXCOR) and a hybrid-capture amplification assay (Digene), versus the widely used branched-DNA (bDNA) assay (Chiron) in the monitoring of HBV DNA levels during antiviral treatment. Serial serum samples from 12 chronically HBV infected patients undergoing a phase II trial of an antiviral drug, 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), were studied. A total of 96 serum samples were tested for HBV DNA using the cross-linking, hybrid-capture amplification, and bDNA assays. In the comparison of the cross-linking and bDNA assays, concordant results were found in 77 (80.3%) samples, no significant difference was found between the median log(10) HBV DNA levels (6.66 versus 7. 17 meq/ml), and the results of the two assays were closely correlated (r = 0.95). In the comparison of the hybrid-capture amplification and bDNA assays, concordant results were found in 79 (82.3%) samples, no significant difference was found between the median log(10) HBV DNA levels (6.98 versus 6.99 meq/ml), and the results of the two assays were closely correlated (r = 0.99). Six (6. 3%) samples by the cross-linking assay and 10 (10.4%) samples by the bDNA assay required retesting because of unacceptably high within-run coefficients of variance. No sample required retesting in the hybrid-capture amplification assay according to the internal validation. In conclusion, the cross-linking and hybrid-capture amplification assays were as sensitive as the bDNA assay for HBV DNA detection and can be recommended for monitoring of HBV DNA levels during antiviral treatment.     

Effects of medication, behavioral, and combined treatments on parents' and children's attributions for the behavior of children with attention-deficit hyperactivity disorder

Seventy-four mothers and 41 fathers and their 6 to 13 year old sons with attention-deficit hyperactivity disorder (ADHD) watched videos of child ADHD symptoms, compliance, and noncompliance. Participants were told either that the child was receiving medication, behavioral treatment, a combination of the two, or was not receiving treatment and were asked to rate the cause of the behavior. Parents attributed less control but greater stability to positive child behaviors when the child was receiving medication. However, for negative behaviors, medication increased attributions of control but diminished stability. With behavior management. compliance was seen as more external and stable and noncompliance as more controllable but less stable. For all treatments, boys reported increased control over ADHD symptoms and noncompliance. The implications of these treatment-related attributions for parenting and children's self-perceptions are discussed.     

T-lymphocyte activation in IgA nephropathy: Serum-soluble interleukin 2 receptor level,interleukin 2 production,and interleukin 2 receptor expression by cultured lymphocytes

The present study was undertaken to examine the T-lymphocyte activation in IgA nephropathy. Serum-soluble interleukin 2 receptor (sIL2R) levels were studied in 29 IgA nephritic patients, 17 patients with chronic glomerulonephritis (non-IgA nephropathy), and 30 healthy controls during an infection-free period. No difference in serum sIL2R level was demonstrated among these three groups of subjects. However, the serum sIL2R levels of IgA nephritic patient rose significantly during clinical exacerbation with synpharyngitic macroscopic hematuria and the serum sIL2R levels fell when hematuria subsided. Mitogen-stimulated cellular interleukin 2 receptor (IL2R) expression, sIL2R release, and interleukin 2 (IL2) production were also examined in peripheral blood mononuclear cells (PBMC) cultured for 24–48 hr in 21 patients with IgA nephropathy, 17 patients with chronic glomerulonephritides, and 17 healthy controls. The total cellular IL2R expression and sIL2R release did not differ among these three groups of subjects. However, the individual T-cell subsets bearing IL2R were distinctly different between IgA nephritic patients and the other two groups of controls. IgA nephritic patients had increased activated CD4+ lymphocytes and reduced activated CD8+ lymphocytes. Furthermore, IL2 production in response to phytohemagglutinin and pokeweed mitogen stimulation was increased in lymphocytes from patients with IgA nephropathy. The IL2 production did not correlate with the quantities of cellular and sIL2R yet the cellular IL2R expression paralleled the sIL2R released by cultured lymphocytes. Our present study suggests that the T lymphocytes from patients with IgA nephropathy have a defect in overproduction of IL2 and increased activated T helper-cell subset upon mitogenic stimulation. Serum measurement of sIL2R could potentially be useful in monitoring the disease activity.     

Novel immunofluorescence assay using recombinant nucleocapsid-spike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a "gold standard" during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chng, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422, 2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.     

Evidence that prostate gonadotropin-releasing hormone receptors mediate an anti-tumourigenic response to analogue therapy in hormone refractory prostate cancer

Gonadotropin-releasing hormone analogue (GnRHa) therapy is an established method of androgen withdrawal in the treatment of prostate cancer. The present study investigated if the expression of prostate GnRH receptors (GnRHRs) might influence the response to GnRHa. GnRHR protein expression was first studied in a panel of prostate cancer cell lines. In androgen-dependent cells, GnRHR expression was unchanged following acute or chronic androgen withdrawal. In these cells, GnRHa significantly inhibited androgen-induced cell proliferation (p = 0.01). In contrast, GnRHa was unable to further suppress basal levels of cell proliferation induced by androgen withdrawal. In androgen-independent prostate cancer cells, variable levels of GnRHR expression were observed. In these cells, GnRHa treatment blocked cell proliferation (p = 0.001) and invasion (up to 70%) induced by fibroblast growth factor stimulation. Crucially, this effect was only evident in cells that expressed high levels of the GnRHR. GnRHa treatment also significantly inhibited the ability of these cells to recover from a cytotoxic insult (50% inhibition). The clinical significance of prostate GnRHR was tested by immunohistochemistry in a preliminary cohort of patients treated with GnRHa or surgical castration. There was no association between GnRHR expression and pathological grade, clinical stage, time to PSA nadir (p = 0.82) (n = 35) or progression to hormone refractory disease (p = 0.22) (n = 21), irrespective of the treatment method. GnRHa therapy in the presence of high GnRHR expression however, was found to be associated with longer disease-specific survival (mean survival 85 months, p = 0.002). In contrast, high GnRHR expression was not associated with survival among surgically castrated patients (mean survival 50 months, p = 0.7). Taken together, these data support the notion of a functional interaction between GnRHa and the GnRHR, which results in an anti-tumourigenic effect on prostate cancer cells. Findings from this report have direct implications for the use of GnRHR as a novel therapeutic target in hormone refractory prostate cancer.