Human herpesvirus type 6 reactivation after haematopoietic stem cell transplantation

Human herpesvirus type 6 (HHV6) is known to reactivate after hematopoetic stem cell transplantation (HSCT) and has been suggested to be associated with increased mortality and severe clinical manifestations, including graft versus host disease (GvHD). The exact etiological role of HHV6 reactivation in increased morbidity and mortality after HSCT remains unclear. This review will focus on the current available evidence of HHV6 reactivation after HSCT and its immuno-modulatory capacities, with particular emphasis on the severe complication GvHD. At present, no effective specific antiviral treatment for HHV6 reactivation has been identified. The currently available antiviral agents are outlined, as well as possible future strategies for the treatment of HHV6 reactivation. Non-toxic, specific treatment or prevention of HHV6 reactivation might improve the safety and efficacy of the HSCT procedure.     

Expression of apoptosis-related proteins and morphological changes in a rat tumor model of human small cell lung cancer prior to and after treatment with radiotherapy,carboplatin, or combined treatment

PURPOSE: In order to understand the apoptosis pathway in tumor cells, differences in cell morphology and expression of apoptosis-related proteins induced by radiation and/or chemotherapy, were investigated in a rat double tumor model comparing cisplatin-sensitive and -resistant human small cell lung cancer tumors. METHODS: The cisplatin-sensitive human small cell lung cancer cell line (GLC4) and its cisplatin-resistant subline (GLC4-CDDP) were each injected subcutaneously in a different hind limb of the rat. After 15-17 days, radiation (10 Gy), carboplatin (25 mg/kg, intraperitoneal), combined treatment, or no treatment was administered. In combined treatment, carboplatin was administered 24 h before radiation. Tumors were removed and fixed 72 h after carboplatin or 48 h after radiation. Paraffin-embedded slides were stained with hematoxylin/eosin for morphology. Expression of the apoptosis-related proteins p53, p21, Rb, bcl-2, bax, c-myc, Fas and Fas-L was assessed immunohistochemically. RESULTS: In the GLC4 tumor, carboplatin treatment increased tumor cell size, tumor cell size heterogeneity, and number of apoptotic tumor cells. After radiation and combined treatment, these changes were more pronounced and multinuclear giant cells were observed. In GLC4-CDDP tumors, minimal changes were detected after carboplatin. Following radiation slight increases in tumor cell size, tumor cell size heterogeneity and number of apoptotic tumor cells were observed. No multinuclear giant cells were seen. After combined treatment, GLC4-CDDP showed cellular changes comparable to those in GLC4 cells after radiation or combined treatment, but no giant cells were observed. Untreated, both tumors were equally positive for p53, bax, Fas, Fas-L, c-myc and negative for Rb. Contrary to GLC4, untreated GLC4-CDDP tumors showed no p21 and bcl-2 expression. After combined treatment, an increase in number of bcl-2 positive cells was found in GLC4-CDDP tumors. No p21 was induced in GLC4-CDDP by any treatment modality. In GLC4 tumors, p21 was induced by radiation alone. No further changes in protein expression were induced by any treatment in GLC4 tumors. CONCLUSION: Following therapy, morphological changes in cell size and cell size heterogeneity were more pronounced in GLC4 than in GLC4-CDDP tumors. However, morphological changes in GLC4-CCDP tumors after treatment indicate that carboplatin plus radiotherapy may be effective also in cisplatin resistant tumor cells. An increase in p21 in GLC4 after radiation might facilitate apoptosis. The increase in number of Bcl-2 positive cells after combined treatment and the consistently negative p21 status after any treatment in GLC4-CDDP may protect these tumor cells from apoptosis as a part of their resistance mechanism to cisplatin.     

Suprabasal p53 immunoexpression is strongly associated with high grade dysplasia and risk for malignant transformation in potentially malignant oral lesions from Northern Ireland

AIMS: No good predictive marker for the malignant transformation of potentially malignant oral lesions (PMOLs) is currently available. This study re-evaluated the value of p53 immunoexpression to predict malignant transformation of PMOLs after discounting possible confounding factors. METHODS: PMOLs from 18 patients who showed progression to carcinoma, 16 of the respective carcinomas, and PMOLs from 18 matched controls were evaluated by immunohistochemistry (IHC) for p53 expression. A mouse monoclonal antibody that detects wild-type and mutant forms of human p53 was used. The p53 immunostaining pattern was also correlated with the degree of dysplasia. RESULTS: Suprabasal p53 staining was significantly associated with high grades of dysplasia (p < 0.01). The specificity and positive predictive value (PPV) for malignant transformation of suprabasal p53 staining were superior to the assessment of dysplasia, but sensitivity was inferior. All carcinomas derived from PMOLs with suprabasal p53 showed strong p53 immunostaining. However, the absence of suprabasal p53 staining and/or dysplastic changes did not preclude malignant transformation in a considerable proportion of PMOLs. CONCLUSIONS: This study confirms and extends previous findings that suprabasal p53 immunoexpression has a high PPV for malignant transformation of PMOLs and can be used as a specific marker for lesions that are at high risk for malignant transformation. The absence of suprabasal p53 staining (that is, absence of, or basal, p53 staining) is non-informative for prognostic purposes. Because of its limited sensitivity, p53 IHC is not a substitute for the assessment of dysplasia in the evaluation of PMOLs. Instead, p53 IHC emerges as a clinically useful supplement of histopathological assessment in the prognosis of PMOLs.     

Rat cytomegalovirus induces cellular purine and pyrimidine nucleoside kinases in rat embryo fibroblasts and TK− rat-2 cells. Correlations with the antiviral activity of acyclovir

Summary Rat cytomegalovirus (RCMV) induces a cytosol thymidine kinase (TK) in G₀-phase rat embryo fibroblasts (REF), but not in a TK deficient rat cell line (R-2), though virus titers in both cell types reached comparable levels. The results indicate that TK is neither virus-coded nor is required for a productive infection in R-2 cells. A deoxycytidine kinase (dCK) is induced in either growing or RCMV-infected REF and R-2 cells, suggesting that dCK is essential for both host-cell and viral DNA synthesis. A deoxy-guanosine kinase (dGK) is detectable in low concentrations in either growing or G₀-phase REF and R-2 cells suggesting that this enzyme is cell-cycle independent. In contrast, RCMV induces high persisting levels of dGK, particularly in R-2 cells, indicating that this enzyme is of crucial importance for viral DNA synthesis. By comparison of thermostabilities and electrophoretic mobilities (R_f for TK, dCK and dGK were 0.12; 0.97; and 0.54, respectively) the enzymes were found to be substrate specific but of cellular origin. In contrast to TK and dCK, only dGK is inhibited by Acyclovir (K_i=320 µm). It is suggested that RCMV inducable dGK is an important enzyme determining thein vitro anti-CMV activity of Acyclovir.With 8 Figures     

ALK expression in extranodal anaplastic large cell lymphoma favours systemic disease with (primary) nodal involvement and a good prognosis and occurs before dissemination

AIMS: In anaplastic large cell lymphoma (ALCL), the site of origin has been described as an important prognostic factor. Recently, a fusion protein containing anaplastic lymphoma kinase (ALK) was described in systemic nodal ALCL, and shown to be associated with a good prognosis. The aims of this study were to investigate whether the presence of ALK protein differs between ALCL of different sites of origin; to determine whether ALK expression occurs before dissemination to other sites; and, finally, to investigate whether the site of origin remains a prognostic parameter in ALK negative ALCL. METHODS: ALK expression, as detected by immunohistochemistry using the monoclonal antibodies ALK1 and ALKc, was studied in 85 ALCLs from different sites of origin. In 22 patients, ALK expression was studied in multiple biopsies from different sites (including 13 skin, 16 lymph node, and nine other). Overall survival time was analysed using the Kaplan Meier method. RESULTS: ALK expression was found in 20 of 51 systemic ALCLs with (primary) nodal involvement. No ALK expression was found in 15 primary cutaneous, 14 gastrointestinal, and five nasal ALCLs. Multiple and subsequent biopsies of patients showed ALK expression to be identical to that seen in the primary diagnostic biopsy. Kaplan Meier survival curves showed that in ALK negative ALCLs originating from different sites, primary cutaneous cases are associated with an excellent overall survival, whereas the other cases show a comparable five years survival of less than 40%. CONCLUSIONS: If present, ALK expression favours systemic ALCL with (primary) nodal involvement, and can be used in differentiating between extranodal involvement of systemic (nodal) ALCL and primary extranodal ALCL. ALK is expressed consistently in multiple biopsies of a given patient, indicating that the chromosomal abnormality leading to aberrant ALK expression occurs before dissemination to other sites. Finally, in ALK negative non-cutaneous ALCLs, different sites of origin show comparable poor survival.     

Accuracy of eosinophils and eosinophil cationic protein to predict steroid improvement in asthma

BACKGROUND: There is a large variability in clinical response to corticosteroid treatment in patients with asthma. Several markers of inflammation like eosinophils and eosinophil cationic protein (ECP), as well as exhaled nitric oxide (NO), are good candidates to predict clinical response. AIM: We wanted to determine whether we could actually predict a favourable response to inhaled corticosteroids in individual patients. METHODS: One hundred and twenty patients with unstable asthma were treated with either prednisolone 30 mg/day, fluticasone propionate 1000 microg/day b.i.d. or fluticasone propionate 250 microg/day b.i.d., both via Diskhaler. They were treated during 2 weeks, in a double-blind, parallel group, double dummy design. We measured eosinophils and ECP in blood and sputum, and exhaled nitric oxide as inflammatory parameters before and after 2 weeks in order to predict the changes in forced expiratory volume in 1 s (FEV1), provocative concentration of methacholine causing a 20% fall in FEV1 (PC20 Mch), and asthma quality of life (QOL). Secondly, to test whether these results were applicable in clinical practice we determined the individual prediction of corticosteroid response. RESULTS: We found that changes in FEV1, PC20 Mch and QOL with corticosteroids were predominantly predicted by their respective baseline value and to a smaller extent by eosinophils in blood or sputum. ECP, measured in blood or sputum, was certainly not better than eosinophils in predicting clinical response to corticosteroids. Smoking status was an additional predictor for change in FEV1, but not for change in PC20 Mch or QOL. Prediction of a good clinical response was poor. For instance, high sputum eosinophils (> or = 3%) correctly predicted an improvement in PC20 Mch in only 65% of the patients. CONCLUSION: Our findings show that baseline values of the clinical parameters used as outcome parameters are the major predictors of clinical response to corticosteroids. Eosinophil percentage in blood or sputum adds to this, whereas ECP provides no additional information. Correct prediction of clinical response in an individual patient, however, remains poor with our currently used clinical and inflammatory parameters.     

High resolution microarray comparative genomic hybridisation analysis using spotted oligonucleotides

BACKGROUND: Currently, comparative genomic hybridisation array (array CGH) is the method of choice for studying genome wide DNA copy number changes. To date, either amplified representations of bacterial artificial chromosomes (BACs)/phage artificial chromosomes (PACs) or cDNAs have been spotted as probes. The production of BAC/PAC and cDNA arrays is time consuming and expensive. AIM: To evaluate the use of spotted 60 mer oligonucleotides (oligos) for array CGH. METHODS: The hybridisation of tumour cell lines with known chromosomal aberrations on to either BAC or oligoarrrays that are mapped to the human genome. RESULTS: Oligo CGH was able to detect amplifications with high accuracy and greater spatial resolution than other currently used array CGH platforms. In addition, single copy number changes could be detected with a resolution comparable to conventional CGH. CONCLUSIONS: Oligos are easy to handle and flexible, because they can be designed for any part of the genome without the need for laborious amplification procedures. The full genome array, containing around 30000 oligos of all genes in the human genome, will represent a big step forward in the analysis of chromosomal copy number changes. Finally, oligoarray CGH can easily be used for any organism with a fully sequenced genome.     

Improved PCR sensitivity for direct genotyping of Chlamydia trachomatis serovars by using a nested PCR.

Successful amplification of omp1 DNA by PCR is crucial in the genotyping of Chlamydia trachomatis when directly performed with clinical samples (J. Lan, J. M. M. Walboomers, R. Roosendaal, G. J. van Doornum, D. M. McLaren, C. J. L. M. Meijer, and A. J. C. van den Brule, J. Clin. Microbiol. 31:1060-1065, 1993). Several primers flanking the four variable domains of the omp1 gene were selected and tested for sensitivity in several nested PCRs with serial dilutions of serovar G. The optimal sensitivity obtained was 0.1 to 0.01 inclusion-forming units, similar to that obtained in the C. trachomatis plasmid PCR. With this approach, any C. trachomatis PCR-positive sample can be typed.     

Induction of cytosolic and microsomal epoxide hydrolases and proliferation of peroxisomes and mitochondria in mouse liver after dietary exposure to p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid

The effects of dietary exposure to 0.125% (w/w) p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid on the content of peroxisomes and levels of certain xenobiotic-metabolizing enzymes in mouse liver have been investigated. In agreement with the literature on rat liver 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were found to cause extensive proliferation of peroxisomes (as judged by the total levels of "mitochondrial" protein, carnitine acetyltransferase, cyanide-insensitive palmitoyl-CoA oxidation and catalase) in mouse liver. On the other hand, exposure to p-chlorophenoxyacetic acid did not significantly affect any of these parameters. As with certain other peroxisome proliferators, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid increased total cytochrome oxidase activity as well. In addition, dietary exposure to 2,4-dichlorophenoxyacetic acid and to 2,4,5-trichlorophenoxyacetic acid resulted in increases in the activities of cytosolic and microsomal epoxide hydrolases in mouse liver and generally less pronounced increases in the total cytosolic glutathione transferase activity and microsomal content of cytochrome P-450. In the case of cytochrome P-450, this process can be said to be a true induction (i.e. the amount of enzyme protein is increased), because the assay procedure for cytochrome P-450 measures holoenzyme amount. Immunoquantitation demonstrated that this was also the case for the changes in cytosolic epoxide hydrolase. The dramatic differences in proliferation of peroxisomes and induction of xenobiotic-metabolizing enzymes seen here with compounds differing relatively little in structure may indicate that a receptor mechanism of some kind is involved.     

Characterization of the induction of cytosolic and microsomal epoxide hydrolases by 2-ethylhexanoic acid in mouse liver

When mice were exposed to 1% 2-ethylhexanoic acid in the diet, cytosolic and microsomal epoxide hydrolase (EC 3.3.2.3) activities were increased maximally (2-2.5- and 0.5-1-fold, respectively) after 3 days. Immunochemical quantitation of these enzymes indicated that the process involved was a true induction in both cases. Maximal levels of peroxisome proliferation (as indicated by carnitine acetyltransferase activity) were obtained after 7 days of exposure. All three of these activities returned to control levels within 4 days after termination of the treatment. The liver somatic index was slightly increased after 4 days of administration of 1% 2-ethylhexanoic acid, but the protein contents of the "mitochondrial," microsomal, and cytosolic fractions were unaffected. The activity of peroxisomal palmitoyl-CoA beta-oxidation was increased 2-fold, whereas peroxisomal catalase activity was unaffected. Exposure to 2-ethylhexanoic acid also increased cytochrome oxidase activity, suggesting an effect on mitochondria. Other parameters of detoxication--i.e. total microsomal cytochrome P-450 content, cytosolic glutathione transferase activity toward 1-chloro-2,4-dinitrobenzene, and the "cytosolic" epoxide hydrolase activity localized in the "mitochondrial" fraction--were not affected by 4 days of treatment with 1% 2-ethylhexanoic acid.