We evaluated whether social deprivation affected decision-making for breast cancer surgery. Of 3419 patients, 53.6% had mastectomy and this was predicted by deprivation, age, tumour size and hospital, all of which retained significance on multivariate analysis, except deprivation. Pathological characteristics and surgical decision-making determined choice of operation not deprivation. Awareness of health inequalities between rich and poor has never been greater. The recent publication of health and life expectancy data in Scotland has shown that the gap between rich and poor in Glasgow persists  status and hospital were individually examined for their association with surgical management using Of the 3570 patients entered onto the database, 3419 had tumours smaller than 5 cm. Of these, 1588 (46.4%) underwent conservation surgery and 1831 (53.6%) mastectomy.On univariate analysis, deprivation, tumour size, nodal status, grade, method of diagnosis and hospital varied significantly with type of surgery .Women from deprived areas were significantly more likely to have larger, symptomatic tumours . There P=0.015), but lost its significance when tumour size was added . The multivariate analysis showed that tumour size, nodal status, histological grade, method of diagnosis and hospital were independently predictive of surgical management  with the lowest uptake in the most deprived groups, while the UK average is 75.5% (data from NHS Breast Screening Programme). Both tumour size and method of diagnosis were independently predictive of mastectomy, so it is likely that tumour size and fewer screen-detected tumours determine surgical management rather than the biased treatment of deprived women. Therefore, to some extent, our mastectomy rate reflects higher levels of deprivation in Glasgow.The populations served by the different hospitals are similar in age and access to radiotherapy services, although their levels of deprivation differ. In the univariate model, the relatively low mastectomy rate in hospitals 1 and 4 is due to their large breast screening practice. However, in the multivariate model, which included method of diagnosis, hospital of treatment was independently predictive of surgical management. This indicates that individual surgeons have an influence over choice of surgical management. Although guidelines have been produced recommending suitability for conservation surgery, there still appears to be a lack of consensus among surgeons.It does appear that women from deprived areas are being treated appropriately and the choice of surgery is based on tumour characteristics. However, the wide variation in mastectomy rate between hospitals suggests a lack of consensus on the best surgical treatment of breast cancer.


E. coli to individually delete each of the genome's 70 unique ORFs. The collection of viral mutants obtained was systematically examined both in MeWo cells and in cultured human fetal skin organ samples. We use our genome-wide deletion library to provide novel functional annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be essential for viral replication. Among the 26 non-essential ORF deletion mutants, eight have discernable growth defects in MeWo. Interestingly, four ORFs were found to be required for viral replication in skin organ cultures, but not in MeWo cells, suggesting their potential roles as skin tropism factors. One of the genes (ORF7) has never been described as a skin tropic factor. The global profiling of the VZV genome gives further insights into the replication and pathogenesis of this virus, which can lead to improved prevention and therapy of chicken pox and shingles.The Varicella Zoster Virus (VZV) is a ubiquitous human alpha-herpesvirus that is the causative agent of chicken pox and shingles. Although an attenuated VZV vaccine (v-Oka) has been widely used in children in the United States, chicken pox outbreaks are still seen, and the shingles vaccine only reduces the risk of shingles by 50%. Therefore, VZV still remains an important public health concern. Knowledge of VZV replication and pathogenesis remains limited due to its highly cell-associated nature in cultured cells, the difficulty of generating recombinant viruses, and VZV's almost exclusive tropism for human cells and tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka) genome into a bacterial artificial chromosome that included a dual-reporter system (GFP and luciferase reporter genes). We used PCR-based mutagenesis and the homologous recombination system in the  The Varicella Zoster Virus (VZV) is the causative agent of chicken pox and shingles. The long-term efficacy of the current chickenpox vaccine is yet to be determined, and the current shingles vaccine fails to provide protective immunity for a substantial number of individuals. Shingles can also lead to post-herpetic neuralgia (PHN), a debilitating condition associated with an intractable pain that can linger for life. Therefore, VZV remains an important public health concern. We use growth-rate analysis of our genome-wide deletion library to determine the essentiality of all known VZV genes, including novel annotations for 51% of the VZV proteome. We also discovered a novel skin-tropic factor encoded by ORF7. Overall, our identification of genes essential for VZV replication and pathogenesis will serve as the basis for multiple in-depth genetic studies of VZV, which can lead to improved prevention and therapy of chicken pox and shingles. For example, essential genes may be appealing drug targets and genes whose deletion causes a substantial growth defect may be prospective candidates for novel live attenuated vaccines. Human varicella-zoster virus (VZV) is a widespread human alpha-herpesvirus, and the majority of the US population has been previously exposed in vivo virulence factors on a large scale VZV is the smallest member of the human herpesvirus family, with a linear double-stranded DNA genome (125 kb) that encodes 70 unique ORFs. As a result of the recent development of a VZV cosmid system and of the severe combined immunodeficient mouse model with xenografts of human tissue (SCID-hu), many viral ORFs have been investigated in both biochemical and functional studies, shedding light upon several VZV gene functions The knowledge of VZV replication and pathogenesis is limited, in part because of its highly cell-associated nature in cultured cells and the difficulty of generating recombinant viruses. In order to circumvent some of these problems, we cloned the VZV (p-Oka strain) genome as a bacterial artificial chromosome (BAC) carrying both green fluorescent protein (GFP) and luciferase reporter genes in vivoHuman fetal skin organ culture (SOC) has been previously established to mimic VZV skin infection, which allows for the study of VZV replication and pathogenesis Luc) was used to individually delete and/or mutate each of the 70 unique ORFs by employing the E. coli DY380 strain recombination system E. coli was transfected into human melanoma (MeWo) cells, and the results provide direct evidence of that 44 ORFs are essential for viral replication in cultured MeWo cells and 26 are non-essential. Moreover, among the non-essential gene group, 8 ORF deletion mutants showed significant growth defects compared to the wild-type strain  using a PCR-based approach E. coliAll VZV ORF deletion mutants were constructed from BAC mutants with a luciferase reporter  were found to be essential in this study. ORF4 and ORF62/71 are incorporated into the viral tegument, and both encode immediate-early (IE) proteins with transcriptional regulatory activity Most of the VZV ORFs that encode glycoproteins are essential. Glycoprotein K (gK) (encoded by ORF5) −4) and for DNA packaging ; ORF28 encodes the catalytic subunit of VZV DNA polymerase and ORF16 encodes the subunit of the viral DNA polymerase processivity factor −3) and unknown  functional categories , and the peak signals of the viral detection assay were at least 5-fold less than were those of the wild-type parental strain  . Accordil strain . Two of In vitro growth curve analysis showed that these ORF deletion mutants had the same growth kinetics as their wild-type parental strain, VZVLuc  model has been proven to be a simple and convenient alternative to the SCID-hu mouse model in the study of VZV pathogenesis −19). Only two of these genes (ORF35 and ORF67) have been previously reported to be required for viral growth in SCID-hu skin mouse models Every deletion mutant that showed severe growth defects in cultured MeWo cells also demonstrated significantly slow growth kinetics in human skin samples when compared to wild-type VZV  Among the 18 VZV ORFs dispensable for viral replication in cultured MeWo cells, 14 were also dispensable for viral replication in skin tissue . Among tin vitro  appear to have selective impacts on viral replication in skin tissue. The growth of each virus in SOC was compared with its growth in cultured MeWo cells. These ORF deletion mutants grew like the wild-type strain in vitro . In conttro −19; . For instro −19; . In skintro −19; . In contin vitroin vivoThree skin-tropic virulence factors  have been previously identified. VZV ORF10 encodes a tegument protein that enhances transactivation of VZV genes, and it was shown to be dispensable for VZV replication th codon from TGT to the TGA stop codon  by mutating the 5odon see . Just liodon see  but had odon see .In this study, a global functional analysis of the entire VZV genome was performed that emphasized the identification of viral ORFs important for viral replication both in cultured MeWo cells and human fetal skin organs. We took full advantage of the highly efficient luciferase VZV BAC system and obtained a library of single ORF deletion mutants. Advanced live culture bioluminescence imaging technology allowed us to systematically test a large number of mutant viruses for comparing viral growth kinetics in different systems.VZV has a 125-kb DNA genome encoding 70 unique open reading frames . In thisIn the current study, we have reported ORF7 as a novel VZV skin-tropic factor. ORF7 encodes a 29-kDa tegument protein, and its function remains unknown. The homolog of the VZV ORF7 protein in the herpes simplex virus is the UL51 protein. Recent studies showed that deletion of HSV-1 UL51 causes reduced size plaque formation and low infectivity Despite the large differences between herpesvirus genomes (ranging from 125 kb to >230 kb), all the herpes viruses studied thus far have a similar number of essential genes. For example, HSV-1 encodes 37 essential genes and 48 non-essential genes Unlike the other functional profiling studies performed on HCMV −4 by t-test). The 10 largest VZV ORFs are all essential, while 8 out of 11 VZV ORFs less than 600 bp are non-essential.There is also an apparent size difference between essential and non-essential ORFs. Essential ORFs are significantly larger in size compared to non-essential ones  yet they also showed that failure to express either of these genes resulted in growth defects All of our results are in agreement with previous VZV functional annotations, except for those on ORFs 9A, 17, 61 and 66, for which we could not generate viral deletion mutant progenies with sufficient titers for growth studies. For example, previous studies indicated that was ORF9A in vivo.Although some studies have shown ORF17 to be dispensable for viral replication in vitro in a previous study ORF61 has also been suggested to be a non-essential gene for viral replication ORF66 has been previously cited as dispensable for viral replication, but we have found it to be essential In this study, we have presented novel functional annotations for 36 VZV genes. Due to the global nature of our study and the lack of well-defined upstream and downstream regulatory regions for most VZV genes, some of our annotations may have to be redefined by more detailed studies  cells were grown in DMEM supplemented with 10% fetal calf serum, 100U of penicillin-streptomycin/ml, and 2.5ug of amphotericin B/ml, as previously described, and used to propagate VZV Luc DNAs were transfected into MeWo cells using the FuGene 6 transfection kit  VZVIn vitro growth curve analyses were carried out by live-cell bioluminescence detection assay. MeWo cells were infected with 100 PFU of infected MeWo cell suspensions on 6-well tissue culture plates. Every 24 h, the cell culture medium was replaced with medium containing 150 ug/ml D-luciferin . After incubation at 37°C for 10 min, the bioluminescent signals were quantified and recorded using an IVIS Imaging System (Xenogen), following the manufacturer's instructions. After each measurement, the luciferin-containing medium was replaced with fresh cell culture medium. Measurements were taken daily from the same plate for 7 days. Bioluminescence signal data from each sample were quantified by manually demarcating regions of interest and analyzed using LivingImage analysis software (Xenogen). It has been demonstrated previously that both the infectious center assay and the luciferase assay correlate well Ex vivo growth curve analyses were carried out by live-tissue bioluminescence assay. Infected MeWo cells were titrated and then re-suspended in skin organ culture media (SOCM). After 24 h of incubation, each skin-tissue section was injected five times with 10 ul of the virus-infected cell suspension  by a 1-ml syringe fitted with a 27-gauge needle attached to a volumetric stepper . After inoculation, the sections were placed individually on 500 um mesh NetWell inserts  that rested above 1ml of SOCM in each well of 12-well plates and followed by a 24 h incubation in a tissue culture incubator, 37°C, 5%CO2. Each 24 h, SOCM was replaced with media containing 150ug/ml of D-luciferin. Following 10 min incubation at 37°C, the bioluminescence being emitted from individual cultured skin-tissue samples was recorded using the IVIS Imaging System. After the measurements, each sample (still on a NetWell insert) was transferred onto new 12-well plates with fresh SOCM. The measuring process was repeated every 24 h for 7 days. Bioluminescence signals from manually defined regions of interest were quantified and analyzed. All experiments were performed in triplicate. Wild-type infections served as positive controls and mock infections served as negative controls.Human fetal skin-tissue samples  were acquired from Advance Biosciences Resources . Skin organ-culture techniques were as previously described 2 test statistic using the mb.long function. P-values for the T2 test statistic were calculated using an F-distribution. The T2 test statistic did an excellent job of quantifying the difference in growth curves, but a very strict p-value cutoff was required in order to define statistically significant growth defects (implying that the test statistic may be too sensitive). Therefore, we used a Mann-Whitney U test in order to determine which individual time points significantly differed between wild type and deletion mutant strains. All strains with reported growth defects have at least 6 significantly reduced time points (p<0.05).Wild type and mutant growth curves  were compared using the “timecourse” Bioconductor package Table S1Sequences of all primers used in VZV genomic functional profiling.(0.08 MB PDF)Click here for additional data file.Table S2Description of Overlapping VZV ORFs.(0.05 MB PDF)Click here for additional data file.Table S3Essentiality of Essential VZV Homologous in HSV and CMV.(0.05 MB PDF)Click here for additional data file.Text S1Supplementary Materials.(0.03 MB DOC)Click here for additional data file.


We investigated the association between VEGF and VEGFR2 expression and the antitumour efficacy of GW654652, in various xenograft models. Statistically significant associations were observed between the antitumour efficacy of GW654652 in xenografts and VEGF protein (P=0.005) and VEGFR2 expression (P=0.041). The oral dose of GW654652 producing 50% inhibition of tumour growth (ED50) decreased with increasing levels of VEGF (r=−0.94); and, in contrast, the ED50 increased with the increased expression of VEGFR2 (r=0.82). These results are consistent with the observed inverse correlation between VEGF and VEGFR2 expression in tumours. These findings support the hypothesis that VEGF and VEGFR2 expression by tumours may predict the therapeutic outcome of VEGFR kinase inhibitors.During the development of indazolylpyrimidines as novel and potent inhibitors of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) tyrosine kinase, we observed that some human tumour xenografts are more sensitive to VEGFR2 kinase inhibitors than others. A better understanding of the basis for this differential response may help to identify a predictive marker that would greatly aid in the identification of a suitable patient population for treatment. One representative compound from the indazolylpyrimidine series is GW654652 that inhibited all three VEGFRs with similar potency. The inhibition of VEGFR2 kinase by GW654652 was about 150 to >8800 more potent than the inhibition of eight other kinases tested. GW654652 inhibited VEGF- and bFGF-induced proliferation in endothelial cells with an IC Vascular endothelial growth factor (VEGF) and its receptors have been implicated in the angiogenesis that is essential for growth and metastasis of solid tumours. Since formation of solid tumours is angiogenesis dependent, several strategies have been developed to inhibit VEGF signal transduction as part of anticancer therapy  of response, and would greatly aid in the identification of a suitable patient population for treatment.VEGFR2 kinase inhibitors are less effective against some tumour xenografts than others -2-methoxyphenyl]-N4-methyl-N4-(3-methyl-1H-indazol-6-yl)pyrimidine-2,4-diamine; GW612286, N4-(3-methyl-1H-indazol-6-yl)-N2-pyrimidine-2,4-diamine; and GW695612X, 4-chloro-3-({4-[methyl(3-methyl-1H-indazol-6-yl)amino]pyrimidin-2-yl}amino)benzenesulphonamide were synthesised at GlaxoSmithKline  , A375P (melanoma), and PC3 (prostate adenocarcinoma) were obtained from the American Type Culture Collection . HN5, human head & neck carcinoma cells, were kindly provided by Helmout Modjtahedi at the Institute of Cancer Research, Surrey, and UK. Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics . GW654652, μl of activated VEGFR kinase  to 10 μl of substrate  and 1 μl of small molecule inhibitor at various concentrations. Plates were incubated at room temperature for 60 min, and the reaction was stopped by the addition of 20 μl 100 mM EDTA. Homogenous time-resolved fluorescence reagents  were added and plates were incubated for a minimum of 10 min. The fluorescence at 665 nm was measured with a Victor plate reader  using a time delay of 50 μs. The data for dose responses were plotted as % inhibition calculated with the data reduction formula: 100 × (1−(U1−C2)/(C1−C2)) vs concentration of compound where U is the unknown value, C1 is the average control value obtained for 1 μl DMSO, and C2 is the average control value obtained for 0.035 M EDTA. Data were fitted with a curve described by: y=((Vmax × x)/(K+x)+Y2), where Vmax is the upper asymptote, Y2 is the Y intercept, and K is the IC50.Vascular endothelial growth factor receptor kinase assays were carried out in a homogenous time-resolved fluorescence (HTRF) format in 384-well microtitre plates using a purified, baculovirus-expressed GST-fusion protein encoding the catalytic carboxyl-terminus of human VEGFR kinase 1, 2, or 3. Reactions were initiated by the addition of 10 Km of the respective substrate. Inhibition of c-Fms, SRC, and Tie-2 was evaluated by an HTRF format and the inhibition of CDK2, CDK4, EGFR, ErbB2, and Eph-B4 was detected by scintillation proximity assay.Inhibition of several other kinases by small molecules was also determined. Each kinase assay was conducted using purified recombinant catalytic domain of the enzyme. The concentration of ATP and kinase-specific biotinylated peptide in each assay was below the apparent 2. The medium was aspirated from the cells, and various concentrations of kinase inhibitors in serum-free medium were added to each well. After 30 min, VEGF (10 ng ml−1) or bFGF (0.3 ng ml−1) was added to the wells. Cells were incubated for an additional 72 h and BrdU (10 μM) was added during the last 18–24 h of incubation. Data were fitted with a curve described by the equation, y=Vmax × (1−(x/(K+x))), where K is equal to the IC50.Effect of kinase inhibitors on cell proliferation was measured using BrdU incorporation method using commercially available kits . Briefly, HUVEC were seeded in a medium containing 5% FBS in type 1 Collagen-coated 96-well plates and incubated overnight at 37°C, 5% CO3, mice were randomised into groups of eight prior to treatment with VEGFR kinase inhibitors. Animals were treated with kinase inhibitors  or vehicle , administered once or twice daily by oral gavage for 2–3 weeks (till the mean tumour volume reached 1000–1500 mm3). Tumour volume was measured twice weekly by calipers, using the formula (length × width × width × 0.5), where length was the longest diameter across the tumour, and width was the corresponding perpendicular. Tumour growth inhibition was calculated by change in the slopes of tumour growth for control and treated tumours. The oral dose of GW654652 producing 50% inhibition of tumour growth  was estimated by a programme that performed a weighted nonlinear regression analysis of data using the equation: y=Vmax(1−(x/(K+x))), where K is equal to ED50. All animal studies were carried out with the appropriate institutional ethical committee approval and they met the standards of both the US federal regulations and those required by the UKCCCR guidelines . When tumours reached a volume of 100–200 mm3) were dissociated into single-cell suspension by enzymatic digestion with DNAse and collagenase. Cells were fixed and permeabilised with LeucoPerm  and labelled for 30 min at 4°C with anti-FLK-1 antibody . Cells were then incubated with goat anti-mouse-biotin antibody followed by streptavidin-FITC. Labelled cells were analysed with an FACSort  and the percentage of VEGFR2-positive cells and channel differences were determined using CellQuest software.Freshly excised tumours . Protein concentrations were determined using a BCA kit, and 120–200 μg of tumour extract was loaded onto a 3–8% Tris-acetate gel. To quantitate the VEGFR2 expressions in lungs, 25 μg of lung extracts  was loaded onto a 3–8% Tris-acetate gel. Proteins were transferred to nitrocellulose membranes. Membranes were blocked for 1 h in TBS  containing 5% (w/v) low-fat milk. Membranes were then probed with a VEGFR2-specific anti-Flk1 antibody , followed by a donkey anti-mouse-HRP antibody. ECL (Amersham) was used for detection, and densitometric analysis of receptor expression was carried out using a BioRad Fluor-S MultiImager). To confirm equal protein loading, membranes were stripped and reprobed with antibody against β-tubulin (Santa Cruz).Snap-frozen tumour xenograft tissues were lysed in RIPA buffer . Samples were analysed by serial dilution and tests were performed at least in duplicates. Vascular endothelial growth factor levels were normalised relative to the protein concentration of the tumour extracts. Circulating human and mouse VEGF levels in plasma were also determined using the same assay.Spearman's nonparametric test was used to investigate the association between quantitatively measured VEGFR2 expression, VEGF expression, and the antitumour efficacy of GW654652.−1  is shown in Figure 1In an effort to identify a suitable VEGFR kinase inhibitor for clinical development, several potent VEGFR2 kinase inhibitors from indazolylpyrimidine series were tested in multiple human tumour xenograft models. Different human tumour xenografts exhibited varying levels of sensitivity to the VEGFR2 kinase inhibitors. Invariably, HN5 and HT29 xenografts showed greater growth inhibition, while A375P and PC3 xenografts demonstrated much lower growth inhibition using multiple compounds at the same dose and schedule. To illustrate this point, antitumour data from three different VEGFR2 kinase inhibitors administered at 30 mg kg50 ranging from 2 to 12 nM (50 ranging from 4 to >10 μM).To further confirm and extend these observations, a more detailed antitumour efficacy study with GW654652 in six different human xenografts was conducted. GW654652 was picked for these studies because of its good pharmacokinetic profile in mouse and dog. GW654652 inhibits all three VEGF receptor kinases with an ICs tested . GW65465−1 dose administered orally on a once day−1 schedule. An oral dose of 10, 30, and 100 mg kg−1 of GW654652 resulted in free Cmax  of 0.06, 0.23, and 1.38 μM, respectively. The plasma concentration remained above the IC50 for VEGF-induced HUVEC proliferation for 0, 2, and 12 h for 10, 30, and 100 mg kg−1 doses, respectively. GW654652 was a potent inhibitor of the HT29, HCT116 and HN5 xenografts with an ED50 ranging from 20 to 28 mg kg−1. However, the inhibition of SW620, PC3, and A375P models was less pronounced with an ED50 ranging from 46 to 114 mg kg−1 dose . In contrast, very high and variable levels of hVEGF in different tumour models were observed. The levels of hVEGF varied from 93 ng mg−1 of A375P tumour protein to 1710 ng mg−1 of HT29 tumour protein .The differential response of the tumours to VEGF/VEGFR antagonists may be due to differences in the levels of either the ligand or the receptor in these tumours. Concentrations of mouse and human VEGF in the tumours and in circulation were measured in the human xenografts. Mouse VEGF levels were very similar in different tumour models and probably are not the major determinants of the differential response to VEGFR2 kinase inhibitors. Median circulating mouse VEGF levels ranged from 59 to 94 pg ml protein . Relativ protein . StatistMd), which represents the magnitude of difference between VEGFR2-specific staining and nonspecific isotype staining. Figure 3BThe VEGFR2 protein expression in tumour xenografts was analysed by flow cytometry using single-cell suspensions from freshly harvested tumour tissue .A comparison of antitumour activity of GW654652 with the VEGFR2 expression revealed that the oral dose of GW654652 producing 50% inhibition of tumour growth in mice increased in rank order with the increasing levels of VEGFR2 in the tumours . A statir=−0.85, P=0.016; in vivo. Since lung tissue contain high amounts of endothelial cell expressing VEGFR2, we looked at the direct effects of recombinant human VEGF121 on the VEGFR2 levels in murine lungs. After the intravenous injection of VEGF121 in mice, the lungs were collected after 5, 10, and 15 min, and the VEGFR2 levels were determined by Western blots. The receptor levels decreased as a function of time in mice injected with VEGF121 compared to untreated (data not shown) or vehicle-treated animals . High levels of VEGF expression alone are capable of initiating angiogenesis in a quiescent tumour vasculature; however, many other factors are also involved in angiogenic switch and maturation of tumour vasculature  protein as a result of gene amplification has been described in tumour cells selected Our present results indicate that tumours producing higher levels of VEGF elicit decreased expression of VEGFR2 and increased sensitivity to a VEGFR2 kinase inhibitor. The inverse relationship between the VEGF expression and the VEGFR2 expression is consistent with the hypothesis that VEGF modulates VEGFR2 by downregulating cell surface expression of VEGFR2 in tumours. These results suggest that VEGFR kinase inhibitors may be more effective in patients with tumours expressing high VEGF and low VEGFR2. Future clinical trials should investigate whether expression of VEGF or VEGFR2 in tumour biopsies from patients receiving VEGFR2 kinase inhibitors can be used as diagnostic markers for VEGF-targeted therapies.


We regret any confusion or inconvenience this error may have caused.In the article "'It Is Our Exercise Family': Experiences of Ethnic Older Adults in a Group-Based Exercise Program," the e-mail address of the corresponding author, Basia Belza, was incorrect. The correct e-mail address is basiab@u.washington.edu. Corrections were made on our Web site on December 19, 2007, and appear online at


The transplantable Guerin epithelioma in Wistar rats was used to test the in vivo effectiveness of 1-(2-hydroxy-3-methoxypropyl)-2-methyl-4-nitroimidazole (P1) as a tumour-cell radiosensitizer after its oral administration at relatively low doses. The radiosensitizing ability of P1 was compared with that of metronidazole. The results indicate that P1 is less toxic than metronidazole, and greater concentrations of P1 in blood and tumour tissues are obtained for the same administered dose of the compounds. The radiosensitizing ability of P1, determined from tumour-regression rates and local-control percentage at 130 days, was higher than that of metronidazole.


The pharmacological interest in lonidamine is related to its ability to enhance the cytotoxic effects of several DNA-damaging anti-tumour agents. This study was undertaken to better understand the in vivo interaction between lonidamine and cisplatin in the treatment of human tumour xenografts, including three carcinoma models characterized by a different responsiveness to cisplatin, in spite of the presence of a wild-type p53 gene in all tumours. The drug combination was more effective in tumour growth inhibition than cisplatin alone against MX-1 breast carcinoma and A2780 ovarian carcinoma, both highly responsive to cisplatin, whereas no influence of ionidamine was observed on anti-tumour activity of cisplatin in the treatment of the relatively resistant IGROV-1 ovarian carcinoma. As cisplatin activity is related to induction of apoptosis, the modulation of drug-induced apoptosis by lonidamine was investigated. Under conditions in which lonidamine itself had negligible effects on tumour growth and apoptosis, the modulating agent stimulated the apoptotic response induced by cisplatin in the responsive but not in the resistant tumours. Tumour response was dependent not only on the drug activation of apoptosis, but mainly on the persistence over time of the event. In the breast carcinoma MX-1, hypersensitive to cisplatin and to the lonidamine+cisplatin combination, the efficacy of drug treatment was associated with phosphorylation of bcl-2 followed by down-regulation of the protein. Lonidamine itself caused a delayed phosphorylation of bcl-2. These results are consistent with the interpretation that lonidamine is effective in modulating biochemical factors involved in regulation of apoptosis.


The VX2 carcinoma has been used extensively as an experimental model for different aspects of tumour behaviour and is usually maintained by serial intramuscular injections of tumour cells. The tumour grows rapidly, infiltrating between muscle bundles into the fibrous tissue replacing ischaemic muscle and into the vascular tree. The most interesting method of spread occurs within the sarcolemma and this may be responsible for the rounded cell nests described in this tumour.


Mice bearing a methylcholanthrene-induced tumour were tested for their cell mediated reactivity to the experimental allergic encephalomyelitis (EAE) peptide of human myelin basic protein (MBP) in the leucocyte adherence inhibition (LAI) test. Tested over a range of peptide concentrations, peritoneal cells (PC) from tumour-bearing mice exhibited optimal adherence inhibition at 640 ng/ml; PC from normal and parasite-infected mice were unreactive. The EAE peptide also stimulated PC from tumour-bearing mice in the E-rosette augmentation (ERA) test and in the macrophage migration inhibition (MMI) test. MMI appeared to be the most sensitive assay, in that significant reaction at peptide concentrations well below those giving significant LAI and ERA. LAI reactivity to the peptide was detected 5 days after tumour transplantation, and continued to be detectable even with very large tumours. In vitro assays were confirmed by demonstration of EAE peptide recognition in vivo, in tumour-bearing and tumour-excised mice, using the delayed-type hypersensitivity reaction. The present experiments demonstrate an antigenic determinant in murine tumours, similar to the well-characterized EAE peptide of human MBP, and establish an animal model for study and characterization of common tumour-associated antigens.


An antigenic activity in pleural effusions of patients with squamous-cell carcinoma of the lung has been prepared in highly purified form by a 5-step fractionation scheme. The purified substance, designated LuCA (lung cancer antigen), was assessed during the course of the fractionation procedure by a radioimmunometric assay carried out with specific soluble reagents. Sensitive saturation-binding assays showed no or only weak uptake of the 125I-labelled antigen preparation by a panel of antisera specific for known bronchogenic tumour markers, and for normal human serum proteins. The preparation appeared to contain lung-tumour-associated antigens, one of them probably distinct for squamous-cell carcinomas. The antigen fraction consists of acid-soluble glycoproteins, and was demonstrated by SDS-polyacrylamide gel electrophoresis as a single band in the mol. wt region of 43,000. The gel-filtration elution volume appeared to indicate the occurrence of the antigenic activity in multiples of this smaller unit. Pilot radioimmunoassays performed with LuCA and an absorbed specific antiserum suggest the possible suitability of the marker preparation for screening lung-cancer patients.


We have studied the relationship between extracellular lactate (LACTe) and extracellular pH (pHe) in murine tumours after vascular occlusion (clamping) followed by reperfusion. In tumours occluded at ambient room temperature, LACTe, measured by microdialysis, increased linearly with time and correlated strongly with the acidification of the extracellular compartment . Significant decrease in LACTe was evident following removal of occlusion at room temperature and is consistent with vascular reperfusion. Occlusion at 35 degrees C, i.e. to maintain tumour temperature during occlusion, resulted in an initial increase in LACTe, which mirrored a rapid reduction in pHe. However further reductions in pHe occurred without increase in LACTe. During vascular occlusion, tumour adenine nucleotide pool decreased and AMP accumulated. AMP subsequently decreased in the 35 degrees C group and this may contribute to the observed differences in accumulation of LACTe, and capacity to recover from vascular occlusion, between the two treatment groups. These data show that extracellular lactate concentration is a good predictor for tumour pH when adequate energy sources are available within the tumour. However, under conditions of more severe stress, resulting in abolition of primary energy stores and cell death, the pHe continues to decline in the absence of a corresponding accumulation of extracellular lactate. This emphasizes the fact that other processes, apart from lactate production, can contribute to reduction in extracellular pH.


The growth of human leukaemia cell lines was also potently inhibited (mean IC 50 = 1.3 μM). Athymic nude mice, carrying s.c. implanted human gastric tumour xenograft GXF251, were treated i.p. with lycobetaine for 4 weeks, resulting in a marked tumour growth delay. Lycobetaine was found to act as a specific topoisomerase IIβ poison. In the presence of calf thymus DNA, pure recombinant human topoisomerase IIβ protein was selectively depleted from SDS-gels, whereas no depletion of topoisomerase IIα protein was observed. In A431 cells immunoband-depletion of topoisomerase IIβ was induced, suggesting stabilization of the covalent catalytic DNA-intermediate in living cells. It is reasonable to assume that this mechanism will cause or at least contribute significantly to the antitumour activity. © 2001 Cancer Research Campaign   http://www.bjcancer.comThe phenanthridine alkaloid lycobetaine is a minor constituent of Amaryllidaceae. Inhibition of cell growth was studied in the clonogenic assay on 21 human tumour xenografts (mean IC


On average, the vascular pO2 in vessels of the upper surface of these tumours is lower than the pO2 of vessels on the fascial side, suggesting that there may be steep vascular longitudinal gradients  that contribute to vascular hypoxia on the upper surface of the tumours. However, we have not previously measured tissue pO2 on both surfaces of these chambers in the same tumour. In this report, we investigated the hypothesis that the anatomical constraint of arteriolar supply from one side of the tumour results in longitudinal gradients in pO2 sufficient in magnitude to create vascular hypoxia in tumours grown in dorsal flap window chambers. Fischer-344 rats had dorsal flap window chambers implanted in the skin fold with simultaneous transplantation of the R3230AC tumour. Tumours were studied at 9–11 days after transplantation, at a diameter of 3–4 mm; the tissue thickness was 200 μm. For magnetic resonance microscopic imaging, gadolinium DTPA bovine serum albumin (BSA-DTPA-Gd) complex was injected i.v., followed by fixation in 10% formalin and removal from the animal. The sample was imaged at 9.4 T, yielding voxel sizes of 40 μm. Intravital microscopy was used to visualize the position and number of arterioles entering window chamber tumour preparations. Phosphorescence life time imaging (PLI) was used to measure vascular pO2. Blue and green light excitations of the upper and lower surfaces of window chambers were made (penetration depth of light ~50 vs >200 μm respectively). Arteriolar input into window chamber tumours was limited to 1 or 2 vessels, and appeared to be constrained to the fascial surface upon which the tumour grows. PLI of the tumour surface indicated greater hypoxia with blue compared with green light excitation (P < 0.03 for 10th and 25th percentiles and for per cent pixels < 10 mmHg). In contrast, illumination of the fascial surface with blue light indicated less hypoxia compared with illumination of the tumour surface (P < 0.05 for 10th and 25th percentiles and for per cent pixels < 10 mmHg). There was no significant difference in pO2 distributions for blue and green light excitation from the fascial surface nor for green light excitation when viewed from either surface. The PLI data demonstrates that the upper surface of the tumour is more hypoxic because blue light excitation yields lower pO2 values than green light excitation. This is further verified in the subset of chambers in which blue light excitation of the fascial surface showed higher pO2 distributions compared with the tumour surface. These results suggest that there are steep longitudinal gradients in vascular pO2 in this tumour model that are created by the limited number and orientation of the arterioles. This contributes to tumour hypoxia. Arteriolar supply is often limited in other tumours as well, suggesting that this may represent another cause for tumour hypoxia. This report is the first direct demonstration that longitudinal oxygen gradients actually lead to hypoxia in tumours. © 1999 Cancer Research CampaignWe previously reported that the arteriolar input in window chamber tumours is limited in number and is constrained to enter the tumour from one surface, and that the


Median patient follow-up was 6.1 years. Single variables as immunohistochemical staining results , histopathological and clinical parameters as well as treatment variables were analysed in order to assess their prognostic importance, in terms of time to recurrence, time to distant metastasis and duration of survival. The extent of tPA tumour cell positivity, categorized as 0–5%, 6–50% and 51–100%, appeared to be of importance for these end-points. Lesions with 51–100% tPA-positive tumour cells were found to have the best prognosis, whereas lesions with 6–50% tPA-positive tumour cells had the worst. Moreover, the prognostic significance of Breslow thickness, microscopic ulceration and sex was confirmed in this study. Multivariate analyses, incorporating these relevant factors, showed that the extent of tPA tumour cell positivity was an independent prognostic factor for distant metastasis-free interval (P= 0.012) and for the duration of survival (P= 0.043). © 2000 CancerResearch CampaignTo investigate whether the course of primary melanoma disease correlates with expression of the various components of the proteolytic plasminogen activation (PA) system, immunohistochemical stainings for activators of plasminogen (tissue type (tPA) and urokinase type (uPA)), inhibitors of plasminogen activation (type 1 (PAI-1) and type 2 (PAI-2)) and the receptor for uPA (uPAR) were performed on 214 routinely processed melanoma lesions. All lesions were primary cutaneous melanomas, minimally 1.5 mm thick, and derived from patients with only local disease at the moment of diagnosis (clinically stage II (T


Flavone acetic acid (FAA) is a compound with proven activity against various transplantable colon cancers in mice. In this study it was evaluated against primary colon tumours, chemically induced by methylazoxymethanol in outbred CF1 mice. FAA was given i.v. at doses of 70 or 100 or 150 mg kg-1 every 7 days for 6 weeks. Only 4 out of 60 FAA treated mice died of toxicity. FAA reduced tumour number and tumour burden compared to control mice (P less than 0.05 at least), with no apparent dose-response relationship. Antitumour activity of FAA was comparable to that of 5-fluorouracil (5-FU) used as standard. Moreover, FAA was more effective that 5-FU against large tumours. FAA levels in plasma and different tissues (including colonic neoplastic lesions) after a single i.v. dose of 150 mg kg-1 were investigated. Tumour FAA levels appear insufficient to be responsible for the antitumour activity based only on a direct FAA cytotoxic effect. The results confirm clinical interest in FAA and suggest that mechanisms other than direct cytotoxicity may be involved in its activity.


S.c. cellular transplants of MS tumours have a high incidence of rejection in adult BALB/c mice, which can then be used as syngeneic regressors. When these tumours were inoculated within a glass cylinder which had been implanted s.c. in BALB/c mice 2 days earlier, 51% of the animals died with progressively growing tumours, compared with 2% in animals which had received the same inoculum directly s.c. This experimental model demonstrates tumour enhancement in a syngeneic system, and duplicates what has been previously reported in two different allogeneic tumour-host combinations, where it was demonstrated that immunological enhancement was operating, since the addition of either progressor serum or soluble tumour antigen significantly increased tumour incidence. For the purpose of investigating whether the glass cylinder model could also modify the immune response of the host to a second tumour challenge, a leukaemia virus known to crossreact with MS was used. Regressors were challenged i.p. with a lethal dose of a leukaemia virus, PLLV. Regressors bearing a glass cylinder showed a 22% survival rate which was significantly lower than that of the s.c. inoculated regressors (71%). This decrease in cross-immunity suggests that the artificially constructed privileged site created by the glass cylinder, by conditioning for tumour enhancement, also decreases immunological memory.


Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma membrane drug transporter P-glycoprotein (P-gp) or the recently discovered multidrug resistance-associated protein (MRP). In this study we investigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123), daunorubicin (DNR) and calcein acetoxymethyl ester  in order to detect the function of the drug transporters P-gp and MRP, using flow cytometry. The effects of modulators on the accumulation and retention of these probes were compared in several pairs of sensitive and P-gp- as well as MRP-overexpressing cell lines. R123, in combination with the modulator PSC833, provided the most sensitive test for detecting P-gp-mediated resistance. Moreover, in a 60 min drug accumulation assay R123 can be regarded as a P-gp-specific probe, since R123 is not very efficiently effluxed by MRP. In contrast to R123, a 60 min DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance. The MRP-specific modulator genistein could be used in combination with DNR, but not with calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells, but is not specific for MRP. Thus, although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR, the former may be used to probe specific MRP activity whereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assays the role and relative importance of P-gp and MRP can be studied in, for example, haematological malignancies.


Hepatocellular carcinoma (HCC) frequently shows an allelic imbalance (AI) on chromosome 16q. In order to define the commonly affected regions on chromosome 16q, we assessed AI studies in 41 HCCs using a panel of 37 microsatellite markers. Thirty-five cases (85%) showed AI at one or more loci. Among the 35 cases with AI, 21 cases showed multiple AI, suggesting the wide scope of deletion on the long arm of chromosome 16, and the remaining 14 cases showed partial AI. Detailed deletion mapping identified two independent commonly deleted regions on this chromosome arm. These included the D16S3106 locus and D16S498 locus. In conclusion, we have demonstrated frequent AI on 16q in HCCs and identified two loci with frequent AI, which may harbour new tumour suppressor genes. © 1999 Cancer Research Campaign


The effect has been examined of various host treatments  on both lung colony formation and clearance of radioactive cells from the lungs after i.v. injection of tumour cells. Two tumour-host models have been used: the non-immunogenic KHT tumour in C3H/Km mice, and the immunogenic EMT6 tumour in BALB/c/Ka mice. Even for the at most weakly immunogenic KHT tumour, the number of artificial pulmonary metastases could be modified by a factor of up to 10(4) by different host treatments before i.v. inoculation of tumour cells. For all pretreatments except immunization, the shape of the curve of loss of radioactivity from the lungs vs time was biphasic, with an initial steep portion representing intravascular death of the tumor cells, followed 1--2 days after tumour-cell injection by a shallow exponential curve. It was concluded that the shallow slope represented spontaneous death of tumour cells in the perivascular tissues. Essentially all the injected tumour cells lodged initially in the lungs, and this was unaffected by the different host treatments. Furthermore, except for specific immunization, cell death in the perivascular tissues was also unaffected by host treatment. However, the survival of the tumour cells during the 24 h after injection (before they became extravascular) was extremely dependent on the particular host pretreatment. It would appear from these studies that host treatments such as C. parvum injection or anticoagulation can markedly affect the number of blood-borne pulmonary metastases, but they will only be effective if given before the tumor cells arrive in the lung vasculature.


The effects of long-term anticoagulation with phenprocoumon on growth of the Lewis lung carcinoma (3LL) were studied. Oral anticoagulation initiated at the day of i.m. transplantation of the 3LL into C57BL mice significantly inhibited primary tumour growth and reduced the number of spontaneous metastases to the lungs. Intermittent anticoagulation was without effect on metastasis formation but still retarded primary growth. There was no influence of anticoagulation on the mean survival time (MST) of tumour-bearing animals. Phenprocoumon appears to improve the results of cyclophosphamide of 5-fluorouracil treatment, but there were no statisticially significant differences. In contrast, bleomycin treatment in combination with adjuvant anticoagulation suggested a possible drug synergy. No significant influence of anticoagulation on the response of the primary tumour to irradiattion was found, though the MST of irradiated and anticoagulated animals was greater than in the solely irradiated controls. The present investigations suggest that coumarin derivatives have some direct tumour-inhibiting capacities, but exert their antimetastatic action via deceleration of the blood clotting mechanism.


Host responsiveness to a progressively growing methylcholanthrene (MC) induced tumour (MC6/2) was studied at varying intervals following subcutaneous (s.c.) tumour implantation by monitoring the in vitro incorporation of tritiated thymidine (3H-TdR) into lymph node cells (LNC) undergoing stimulation in vivo and concurrently determining the total numbers of the lymphoid cells present in these organs at each of the time intervals. It was found that an initial period of rapidly increasing stimulation of DNA synthesis in lymph nodes was soon followed by the onset of a stage of decrease of this activity. Within limits, the larger the tumour inoculum the stronger the initial response. The suppression of stimulation of DNA synthesis that ensued appeared to be directly related to the tumour mass and to the dose of tumour cells implanted. The total numbers of the cells accumulating in nodes also increased initially but remained elevated during the subsequent period of tumour growth. Continued presence of the tumour was essential for the increased DNA synthesis in lymph nodes since tumour removal leads to a rapid decrease to levels found in tumour-free animals. These findings demonstrate that the failure to eradicate an antigenic tumour by its host may not be solely due to "desensitizing" and "blocking" factors but that other important mechanisms are also involved. We suggest that the inability to reject the tumour in this situation is dependent in considerable measure on the development of a state of hyporeactivity in the host due to the partial inhibition of the DNA synthetic response, possibly in T cells of the tumour host, due to "suppressor factor(s)" interacting with the immunocompetent cells.


Effective radioimmunotherapy is limited by slow antibody clearance from the circulation, which results in low tumour to blood ratios and restricts the dose of radiolabelled anti-tumour antibody that can be safely administrated. Avidin and streptavidin clearing agents have been shown to effectively complex and clear radioactive biotinylated antibodies from the circulation, but their immunogenicity may limit their repeated use. We have investigated whether polyethylene glycol (PEG) modification can reduce the immunogenicity of our galactosylated streptavidin  clearing agent without altering its effectiveness as a clearing agent. The immune response evoked in mice after intraperitoneal infection of 30 micrograms of gal-streptavidin was decreased after PEG modification, as shown by lower antibody titres and a reduction in the number of mice that elicited an anti-gal-streptavidin response. The effect of PEG-modified gal-streptavidin on the blood clearance and tumour localisation of a 125I-labelled biotinylated anti-CEA was investigated in the LS174T human colon carcinoma xenograft in nude mice. Although PEG modified gal-streptavidin bound the [125I]biotinylated antibody in vivo, effective clearance from the circulation was inhibited, resulting in very little reduction in the levels of circulation radioactivity, together with a decrease in the antibody localised to the tumour.


Photodynamic therapy is a potential treatment for superficial bladder cancer that utilizes photosensitizer drugs, which are activated by light to cause tissue destruction. However, first-generation photosensitizers cause prolonged phototoxicity, have poor tumour specificity and can accumulate within detrusor muscle, resulting in permanent loss of bladder capacity following treatment. A newer drug, called 5-aminolaevulinic acid (ALA), generates a sensitizer called protoporphyrin IX (PpIX) in situ and has been shown, qualitatively, to be more tumour specific. The fluorescence kinetics of ALA-induced PpIX was investigated in patient biopsies of bladder tumour, normal urothelium and detrusor muscle, both in vitro after incubation of specimens in ALA-rich culture medium for various times and in vivo after instillation of intravesical ALA before endoscopic resection. The fluorescence in tumour tissue was twice that of normal urothelium in vitro and up to tenfold in vivo. There was little ALA-induced fluorescence in detrusor muscle, both in vitro and in vivo. Most importantly, no patients experienced phototoxicity or other adverse events following intravesical instillation of ALA.


Blood lymphocytes from 47 patients with lung carcinoma have been tested for cytotoxicity against cells isolated from the autologous tumour. Significant cytotoxic potential was found in 15 cases. The effectors were also tested against allogeneic tumour targets from lung and other sites. Reactions were only rarely detected (2/32 positive against lung and 1/13 positive against non-lung cells). The restriction of cytotoxicity to the autologous combination was also apparent in in vitro-generated effectors. Blood lymphocytes were co-cultivated with autologous tumour and subsequently tested against autologous or allogeneic targets. Cytotoxicity was found in 13/17 lung tumours against autologous tumour, with no reactions recorded against allogeneic tumour targets, but one case positive against the K562 cell line. These data suggest either the expression of individually distinct antigens on human pulmonary neoplasms, or the requirement for histocompatibility between target and effector in cytotoxicity reactions in man, and therefore differ from previously described patterns of lymphocytotoxicity against human tumours.


Tumour factors therefore appear unlikely to account for socioeconomic gradients in survival.Analysis of population-based registry data ( Colorectal cancer survival is greater in more affluent UK patients . The rolet al, 1989; Known tumour factors associated with a poor outcome in colon cancer are mucin-producing adenocarcinoma (MPA) sub-type, proximal subsite and high tumour grade (poorly differentiated tumours) . Of the The Merseyside and Cheshire Cancer Registry database incorporates information directly extracted from pathology reports and clinical case notes. Data were obtained for all colon cancer cases during 1989–1996. The overall registry data quality is high and compares favourably with other registries . Cases wet al, 1997; et al, 2003) and due to lack of individual-level information. Quintile groups were defined, calculated for England and Wales (1991 census). The first group is the least deprived (taken as the reference group) and the fifth group the most deprived.Socioeconomic status is commonly measured directly  or indirectly by using area-based measures  , in commAge group and histopathological subtype were categorised as shown in Table 12 test for trend. Binary logistic regression models were used to examine the likelihood of MPA tumour subtype (model 1), proximal subsite (model 2) and poor grade (model 3), respectively, by deprivation group, adjusting for sex and age group. Subsite was also adjusted for in model 1 and subtype in models 2 and 3.‘Unknown’ status for tumour subsite, type, grade and diagnosis based on histology was tested for association with deprivation status using the χCases for which the independent variable was unknown were excluded. Cases for which dependent variables were unknown were included in all models, treated as a separate category. The test for trend in the effect of SES was based on regression analysis with Carstairs' score entered as a continuous variable (range from −5 to 15). The Odds ratio (ORs) derived by the test for trend indicates the effect of one unit change in deprivation status on the probability of the outcome under examination.2 for trend P-values: 0.54, 0.67, 0.53 and 0.67, respectively).There were 7393 cases, of which 461(6.3%) were excluded . Most exHaving adjusted for sex, age group and subsite, MPA subtype was less likely in more deprived patients  patients are more likely to have tumours of MPA subtype and proximal subsite.It is possible that there has been a degree of under-ascertainment of MPA subtype in the study dataset −6.4% compared with about 13% reported in US studies . Based on present evidence therefore, it is unlikely that tumour factors could account for observed SE differences in colon cancer survival. Future research should concentrate on the potential influence of differences in healthcare and patient factors.In common with previous UK research in this field, SES was measured ecologically. The theoretical possibility of a true effect of SES on colon cancer tumour factors not having been detected due to misclassification error is acknowledged. Similarly, examined tumour factors might have been unsuitable to explore the study hypothesis and other tumour characteristics, such as vascular invasion and newer biochemical and genomic markers might have been more suitable. However, most ‘newer’ markers are correlated with ‘conventional’ tumour factors such as the ones examined here, making it likely that their SE distributions would also be similar. It was not possible to examine the influence of tumour stage; however, stage


Thirty-one cervical biopsies of invasive carcinoma have been studied by immunohistochemical means using the monoclonal antibody Ki67 to determine tumour cell proliferation rates. A wide range (10-50%) in the extent of Ki67 staining (expressed as the percentage of labelled tumour cells) was observed indicating considerable variation on tumour growth rates. There was no significant relationship between the percentage of positive cells and conventional histological parameters such as cell type or tumour differentiation. Immunostaining with monoclonal antibody Ki67 therefore provides a new approach to the assessment of cervical tumour biopsies which will require long term clinical follow-up to establish its prognostic significance.


One hundred resected cases of squamous cell carcinomas of the oesophagus were reviewed and a series of histological criteria related to the survival time. Two histological features were important in the assessment of survival. Good prognostic factors were a marked lymphocytic response to the tumour and a lack of intravenous tumour infiltration. Presence of tumour in the middle third of the oesophagus, infiltration through the muscularis propria, severe tumour necrosis, glandular or small cell tumour differentiation, lymphatic invasion and lack of peritumoural fibrosis were all factors which tended to worsen prognosis. None of these factors reached statistical significance. The degree of squamous differentiation had no effect on survival.


Good immunoprotection was afforded by A9/SEWA somatic hybrid cells in the C3H mouse/C3H Py tumour system, confirming results previously obtained in the A.SW mouse/SEWA tumour system. However, in this study the immunogenicity decreased with increasing serial subculture of the hybrid line and concomitant chromosome loss.


The exposure of cells to O6-benzyl-N2-acetylguanosine (BNAG) and several guanine derivatives is known to reduce the activity of O6-alkylguanine-DNA alkyltransferase (MGMT) and to enhance the sensitivity of Mer+ (methyl enzyme repair positive) tumour cells to chloroethylnitrosoureas (CENUs) in vitro and in vivo. High water solubility and the pharmacokinetic properties of BNAG make it a candidate for simultaneous administration with CENUs by the i.v. route in human clinical use. In vivo we have shown previously that BNAG significantly increases the efficiency of N'-[2-chloroethyl]-N-[2-(methylsulphonyl)ethyl]-N'-nitrosourea (cystemustine) against M4Beu melanoma cells (Mer+) through its cytostatic activity by the i.p. route, but also increases its toxicity. To investigate the toxicity of BNAG and cystemustine when administered simultaneously in mice, we compared the maximum tolerated dose and LD50 doses of cystemustine alone or in combination with 40 mg kg(-1) BNAG by the i.p. route. The toxicity of cystemustine was enhanced by a factor of almost 1.44 when combined with BNAG. To compare the therapeutic index of cystemustine alone and the cystemustine/BNAG combination, pharmacological tests were carried out in nude mice bearing Mer+ M4Beu human melanoma cells. Isotoxic doses were calculated using the 1.44 ratio. The treatments were administered three times by the i.v. route on days 1, 5 and 9 after s.c. inoculation of tumour cells. Although the toxicities of the treatments were equal, BNAG strongly enhanced tumour growth inhibition. These results demonstrate the increase of the therapeutic index of cystemustine by BNAG and justify the use of BNAG to enhance nitrosourea efficiency in vivo by i.v. co-injection.


Prognostic factors have been studied in 59 men with clinical Stage I non-seminomatous germ-cell tumours of the testis (NSGCTT) seen at the Royal Marsden Hospital between 1973 and 1978. Fourteen of the patients relapsed, and 45 have remained continuously disease-free. Two factors were identified which showed a significant correlation with relapse following radiotherapy: local extent of the primary tumour, and rate of decline of serum alpha-foetoprotein (AFP) and beta-human chorionic gonadotrophin (hCG) levels following orchidectomy. High serum marker levels at the time of referral after orchidectomy were not prognostically significant per se. The presence of tissue-associated hCG in the primary tumour was not prognostically significant. The results were compared with histology and pathological stage of the primary tumour in patients presenting with lung metastases but no clinical evidence of lymph-node disease. Embryonal carcinoma was more commonly associated with a locally invasive primary tumour and with extralymphatic spread than was teratocarcinoma.


An investigation was made into the effect of Corynebacterium parvum therapy on cerebral tumours in mice. I.v. C. parvum caused a slight but significant increase in the survival of BALB/c mice injected intracerebrally (i.c.) with not more than 50 Meth A cells. C. parvum was most effective if given on the same day or 5 days after tumour. If this interval was increased there was no effect. Multiple i.v. injections were no more effective than a single dose. I.v. C. parvum had no influence on the survival of C57BL mice injected i.c. with Lewis tumour cells, and had little effect on the induction of i.c. or s.c. tumours by methylcholanthrene. It was concluded that C. parvum therapy was of little use in the treatment of cerebral tumour in mice. The clinical implications of these findings are discussed.


The vascular effect was determined by laser Doppler flowmetry, power Doppler ultrasonographic imaging and Patent blue staining. The extent of tumour hypoxia was determined immunohistochemically by hypoxia marker pimonidazole and partial pressure of oxygen (pO2) in tumours by electron paramagnetic resonance oximetry. Electrochemotherapy with BLM induced good antitumour effect with 22 days, tumour growth delay and 38% tumour cures. The application of electric pulses to the tumours induced instant but transient tumour blood flow reduction (for 70%) that was recovered in 24 h. During this tumour blood flow reduction, we determined an increase in hypoxic tumour area for up to 30%, which was also reflected in reduced tumour oxygenation (for 70%). According to the described mathematical model, endothelial cells lining in tumour blood vessels are exposed to a ∼40% higher electric field than the surrounding tumour cells, and therefore easily electroporated, allowing access of high BLM concentration to the cytosol. Consequently, electrochemotherapy has, besides the immediate vascular disrupting action, also a delayed one (after 24 h), as a consequence of endothelial cell swelling and apoptosis demonstrated by extensive tumour necrosis, tumour hypoxia, prolonged reduction of tumour blood flow and significant tumour growth delay, and tumour cures. Our results demonstrate that in addition to the well-established direct cytotoxic effect on tumour cells, electrochemotherapy also has an indirect vascular disrupting action resulting altogether in extensive tumour cell necrosis leading to complete regression of tumours.Electrochemotherapy has a direct cytotoxic effect on tumour cells, and presumably, a vascular disrupting effect. In this study, on the basis of the prediction of the mathematical model, histological evaluation and physiological measurements of the tumours were carried out to confirm that electroporation and electrochemotherapy of tumours have a vascular disrupting action. In the study, SA-1 solid subcutaneous sarcoma tumours in A/J mice were treated by bleomycin (BLM) given intravenously (1 mg kg In vivo electroporation has found its place as a drug delivery system for chemotherapeutics, that is electrochemotherapy, and is currently under intensive investigation for delivery of therapeutic genes to normal tissues and tumours, that is gene electrotransfer  cells were used in the experiments. A/J mice of both sexes, purchased from the Institute of Pathology, Faculty of Medicine, University of Ljubljana, Slovenia, were used for this study. They were kept at a constant room temperature (22°C) with a natural day/night light cycle in a conventional animal colony with food and water 5 SA-1 cells in 0.1 ml 0.9% NaCl solution. Six to 8 days after implantation, when tumours reached approximately 40 mm3 in volume, the mice were randomly divided into experimental groups. The study was approved by the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia (No. 323-02-237/01) in compliance with the Guide for the Care and Use of Laboratory Animals .Tumour cells were obtained from the ascitic form of the tumours in mice. Solid subcutaneous tumours, located dorsolaterally in mice, were initiated by an injection of 5 × 10−1 (∼20 μg per mouse) was injected intravenously. Injection volume was 7.5 ml per kg body weight. Eight square wave electric pulses in two sets of four pulses, in two mutually perpendicular directions of 1040 V (at voltage to distance ratio 1300 V cm−1) and with a pulse duration of 100 μs and repetition frequency of 1 Hz, were delivered by two flat, parallel stainless-steel electrodes 8 mm apart , which were placed percutaneously at the opposite margins of the tumour. Good contact between the electrodes and the skin was assured by means of a conductive gel. Electric pulses were generated by a Jouan GHT 1287 electroporator . In the electrochemotherapy protocol, the mice were treated with electric pulses 3 min after BLM injection. All treatments except in the case of electron paramagnetic resonance (EPR) oximetry, laser Doppler measurements and power Doppler ultrasonographic imaging (see below), were performed without anaesthesia and were well tolerated by the animals.Bleomycin  at a dose of 1 mg kge1, e2 and e3) with a Vernier calliper three times per week. Tumour volumes were calculated according to the formula for the volume of an ellipsoid, V=πe1e2e3 × 6−1. The AM (arithmetic mean) of tumour volumes and s.e. were calculated for each experimental group. Tumour doubling time was determined for each individual tumour from the growth curves. Tumour growth delay was calculated for each individual tumour by subtracting the doubling time of each tumour from the mean doubling time of the control group and then averaged for each experimental group. Animals that were tumour free for 100 days were declared cured.Tumour growth was followed by measuring three mutually orthogonal tumour diameters  was injected intraperitoneally. Tumours (3–9 per group) were excised 90 min thereafter, fixed in 10% buffered neutral formalin, embedded in paraffin, cut at three different levels throughout the tumour and stained for pimonidazole according to the manufacturer's instructions. Briefly, the sections were deparaffinised with xylene and rehydrated in ethanol. Sections were exposed to 3% H2O2 in methanol for 5 min to quench endogenous peroxidase activity. Antigen retrieval was achieved by incubating tissue sections with 0.01% pronase for 25 min at 40°C. Sections were then incubated with 1 : 50 dilution of monoclonal anti-pimonidazole antibody  for 40 min at room temperature. This step was followed by biotin-conjugated rabbit anti-mouse secondary antibody and peroxidase-conjugated avidin–biotin complex . Finally, sections were incubated with 3,3′-diaminobenzidine peroxidase substrate and stained with haematoxylin. The slide in which primary antibody was omitted served as a negative control. The mean percentage of area positive for pimonidazole adducts was determined by the image-based analysis system LUCIA . The results of the percentages of necrotic tumour area and the percentages of pimonidazole-positive area were presented as AM and s.e. for each experimental group.The hypoxia marker pimonidazole   that range from 0.1 to 0.5 S m−1  different therapies, grey-scale ultrasound (US) of tumours was performed. The longest and the shortest diameter of tumour and the longest diameter of an echogenic central part (necrosis) were measured. After baseline, US scan evaluation power Doppler US was performed at each time point to check vascularisation. Before microbubble contrast agent  injection, the persistence of the image display on the US machine was set to zero, the signal gain was registered below the noise threshold, and one focus was positioned below the level of the lesion. SonoVue is a sulphur hexafluoride-filled microbubble contrast agent that is licensed for use in abdominal and vascular imaging in most European countries. This agent has a strong nonlinear harmonic response when it is insonated with low acoustic power. The safety and effectiveness of this agent in vascular and parenchymal diagnostic applications have been proved in preliminary experimental and clinical investigations .−1) was mixed with a solution of sodium chloride 0.9% w v−1 solution to form a dispersion containing millions of tiny bubbles of sulphur hexafluoride gas. The solution was injected intravenously (0.2 ml) in mice by using a 27-gauge needle. After 5–10 s, we observed strong peripheral enhancement, and central necrosis was better depicted as on baseline US image. If contrast enhancement was considered inadequate by the on-site examinator after the first bolus injection, the mouse received an additional microbubble contrast agent bolus (0.2 ml). We monitored contrast enhancement for at least 5 min after injection. During the measurements, the mice were anaesthetised in the same way as for EPR oximetry measurements. All US examinations were performed by one experienced radiologist on a US machine  with a 12 MHz, 38-mm long linear transducer. The scanning plane was selected for optimal visualisation of enhancement. Dynamic US data were stored in the US unit until the signal enhancement had completely diminished, usually within 5 min. Static images were obtained before and every 15 s after injection of the contrast agent. The whole examination was stored on a CD for further analysis.SonoVue . The signals were sampled and stored at a frequency of 100 Hz. The theory and applications of laser Doppler flowmetry (LDF) are well known ; brieflyμm) to assess the perfusion inside the tumour (−1) at 3.0 and 1.8% concentrations, respectively, for induction and maintenance of anaesthesia. Although anaesthetised, the mice were kept on an automatically regulated heating pad to prevent hypothermia. Rectal temperature was kept as close as possible to 37°C with variations of up to 0.5°C during single measurements and with the contact surface temperature of the heating pad always below 39°C.For skin measurements, LDF can be used entirely noninvasively. In our study, we, however, used thin invasive probes , thus resulting in a smoothed and downsampled (1 Hz) version of the raw signal, which represented the actual baseline blood flow level. We defined the moment 1 sec before the application of the first electric pulse (3 min after injection) as time zero . From the filtered signal, average blood flow values at distinct predefined time moments before, at and after the treatment were calculated. In general, these time moments were spaced at intervals of 5 min except around time zero, where they were distributed more densely to track rapid changes in baseline blood flow levels occurring after the treatment. Average blood flow values were calculated from filtered signal segments of 20 s length cantered at the aforementioned time moments. As the last step, all average blood flow values were normalised with respect to the average blood flow level measured 5 min before time zero . Thus, the blood flow level in each tumour was expressed as a percentage of the pretreatment blood flow level. These relative values were then used to present the final results and to evaluate the effects of different treatments on tumour blood flow.r=0.962) was found between both methods  was used to estimate tumour perfusion. Patent blue (1.25%) diluted in 0.2 ml 0.9% NaCl was injected at different time points after treatment into the tail vein of animals with tumours from the control, electric pulses, BLM and electrochemotherapy groups. After the dye was distributed evenly through the tissue (1 min), animals were killed and the tumours carefully excised. The excised tumours were removed from the skin, cut in half along their largest diameter and photographed. The images were evaluated using Image J software. The stained area of tumour cross-section was used as an indicator of tumour perfusion. In the study comparing Patent blue staining and the pharmacological method measuring the relative tumour blood flow, a good correlation ( methods .2) in tumours and subcutaneous tissue on the contralateral side before and after treatment of tumours with electric pulses, BLM and electrochemotherapy. Electron paramagnetic resonance oximetry is a non-invasive method (after insertion of the paramagnetic probe), which allows monitoring of pO2 repeatedly at the same point in the tissue over long periods of time  from Bubinga tree , which is sensitive to oxygen, was implanted into the tumour centre and periphery (tumour volume approximately 40 mm3) and in the subcutis 1 day before the treatments. Its EPR spectral linewidth was measured at different time points up to 2 days after the treatment. The char implantation with a sterile needle did not provoke bleeding. In the presence of oxygen, the linewidth of the EPR lines is broadened; the extent of broadening depends on pO2 (2 in the region in contact with the probe was measured.Electron paramagnetic resonance (EPR) oximetry was used to evaluate changes in the partial pressure of oxygen , both designed by Professor T Walczak . Typical spectrometer settings were modulation frequency, 100 kHz; modulation amplitude not more than one-third of the peak-to-peak linewidth, and scan range, 2 mT. The linewidth of the EPR spectra reflects the pOFor EPR oximetry, animals were anaesthetised with a mixture of Domitor  and 10% ketamine  administered intraperitoneally. During anaesthesia, body temperature was kept at physiological values by a regulated heating pad on which the mice were placed .t-test for individual pairwise comparisons, with values of P<0.05 considered as significant. The SigmaStat statistical software was used for statistical analysis .Significance tests were carried out on the data groups using analysis of variance followed by the Bonferroni Electrochemotherapy with BLM was very effective in treatment of subcutaneous SA-1 tumours, resulting in substantial tumour growth delay and high percentage of tumour cures, compared to untreated tumours and tumours treated with BLM or application of electric pulses alone .P<0.05 compared to other treatment groups). The necrotic areas were found mostly in the centre of the tumours and consisted of cells with eosinophilic cytoplasm and pycnotic nuclei. In some cells, acute cellular swelling and nuclear disappearance with cell ghosts were also found. Central necrosis was surrounded by a thin viable tumour rim that led to regrowth after 8–10 days in those tumours that were not cured (P<0.05) in the extent of tumour necrosis up to 24 h after the treatment confirmed the previous observation that the application of electric pulses alone to the tumours induced a small but insignificant retardation in tumour growth .Electrochemotherapy with BLM had, as already mentioned, significant antitumour effect on tumour growth. We specifically observed significant increase in extent in tumour necrosis reaching ∼50% at 48 h after the treatment  for the vessel perpendicular to the field direction and for the vessel tilted by 30° and 60° with respect to the field direction. As the plots show, the electric field in the tissue at the very edge of the lumen, that is in the endothelium of the blood vessel, can exceed the homogeneous value by as much as 42% in the extreme case  and by 10–30% in typical cases, represented by different orientations of the vessels. This is the consequence of the significantly higher conductivity of the blood with respect to the surrounding tissue , due to which the electric current is locally concentrated in the vicinity and inside the blood vessel. In the blood, the electric field is only 60–90% of the homogeneous field in the tissue, which reflects the fact that the electric field is the ratio between the current density and the conductivity and that the conductivity of blood is considerably higher than the conductivity of the surrounding tissue. Consequently, the model predicts that endothelial cells lining the blood vessels are exposed to up to 40% higher electric field than the surrounding tumour cells.The local distortion of the electric field in the vicinity of a blood vessel is illustrated in the two plots in diameter  and for diameter . DenotinWe have already demonstrated in previous studies that electric pulses and electrochemotherapy significantly reduce the blood flow of the treated tumours . In thisWe were further able to follow the exact profile of microcirculatory changes by means of LDF after application of electric pulses to the tumours or electrochemotherapy in a small volume of tumour tissue. P<0.001 for all time instants after time zero in P<0.001) between control and BLM alone compared to the group of tumours that was exposed to electric pulses or the electrochemotherapy group. Differences between the groups before and at time zero in P>0.05). It was also interesting to note that the two groups involving the use of BLM  developed higher blood flow levels after treatment than the corresponding two groups without BLM , which can be observed in P>0.05).Tumour perfusion changes were also measured by Patent blue staining of the tumours with similar vascular effects as observed with other methods of measurement . An immeBlood flow measurements showed that there is an immediate reduction of tumour blood flow after the application of electric pulses and electrochemotherapy. Owing to the effect of applied electric pulses on the vascular supply of the tumours, the extent of hypoxic regions in the tumours as a consequence of reduced tumour blood flow was determined.Tumour sections were immunohistochemically stained for protein adduct of reductively activated pimonidazole. Positive staining was found in the cytoplasm of tumour cells and some positive nuclei of tumour cells were also determined. As pimonidazole is known to be preferentially bound by hypoxic tumour cells, the detection of pimonidazole adducts using monoclonal antibodies can serve as a method for measuring tumour hypoxia. In most control tumours, discrete foci of pimonidazole positivity were found. In BLM-treated tumours, a rim of positive cells was found mainly around the necrotic area. In electric pulses and in the electrochemotherapy-treated group of tumours, reticular staining patterns were observed at some distance from blood vessels, with some diffuse pimonidazole-positive area. In some slides from the electrochemotherapy-treated group of tumours, the endothelial cells of blood vessels were also positive. The tumour cells around these pimonidazole-labelled vessels did not show pimonidazole immunoreactivity .The percentage of pimonidazole-positive tumour areas in viable tumour tissue was determined and the results are presented in 2 values were recorded in the tumours that were treated, as well as in the subcutaneous tissue that was not treated and was located on the contralateral side from the tumour. The pO2 values in the subcutaneous tissue were higher than those measured in the tumours. Furthermore, the pO2 values in the centre of the tumour were lower than those in the periphery of the tumours  than the application of electric pulses alone (for 8%).All treatments, even BLM alone, had a rapid effect on pOreatment . We alsoreatment . The sysThe results of our study demonstrate that besides its direct cytotoxic effect on tumour cells, electrochemotherapy with BLM also has a vascular disrupting action by causing a rapid shutdown of tumour blood flow, leading to reduced tumour oxygenation, increased tumour hypoxia and extensive tumour cell necrosis. Furthermore, owing to the blood vessel changes caused by a high concentration of BLM in the endothelial cells of tumour blood vessels, this therapy has prolonged vascular disrupting effects leading to pronounced antitumour effectiveness. The mathematical model of electric field distribution in and the around tumour blood vessel supports the physiological data by demonstrating that endothelial cells are exposed to a ∼40% higher electric field compared to the tumour tissue farther from the blood vessel.in vivo has great potential as a drug delivery system, as it is universal and easily applicable, also in humans. It can be used for the delivery of various molecules that have a hampered access to cytosol, such as some widely used chemotherapeutic drugs like BLM and cisplatin, or naked DNA  and delayed effect due to the exposure of these cells to a high concentration of BLM causing endothelial cell death as observed in histological specimens. The first phenomenon is present up to 8–12 h after electroporation of the tissue and is due to disruption of endothelial cell lining in tumour vessels leading to some extent of tumour cell death present up to 24 h, but with quick recovery without significant antitumour effect. The second phenomenon is due to endothelial cell death caused by electrochemotherapy that in addition to the vascular disrupting action of electroporation induces a prolonged shutdown of tumour blood flow and consequently a cascade of tumour cell death in the surrounding tissue that becomes evident at longer times after therapy. Further support for this second phenomenon is the observation that predominantly central tumour necrosis after electrochemotherapy was observed, which is characteristic for vascular disrupting agents.Several two- and three-dimensional numerical calculations have demonstrated that an adequate distribution of electric field above the threshold value for electroporation is important for effective electrochemotherapy . By applElectroporation compared to other vascular disrupting agents such as CA-4-P and DMXAA, which have specific effects on tumour blood vessels as opposed to normal blood vessels, is not specific and selectivity is achieved by the local application of electric pulses to the tumours. The vascular disrupting effects of vascular disrupting agents on tumour endothelial cells involve the rapid reorganisation of the actin cytoskeleton, which is mediated by the disruption of the tubulin cytoskeleton for CA-4-P, but not DMXAA . The actElectroporation, similar to vascular disrupting agents, induces a very rapid and significant reduction in tumour blood flow, which can be detected already within a minute after the application of the treatment. The proposed mechanism for rapid tumour vascular shutdown after treatment with CA-4-P or DMXAA involves the disruption of the cytoskeleton of endothelial cells, leading to cell shape changes and an increase in the permeability of the cell monolayer. This would increase vascular resistance to blood flow. Apoptosis of endothelial cells might also contribute to vascular shutdown. Other contributing factors are platelet activation and serotonin release leading to active vasoconstriction. The protein leakage due to the increased tumour vascular permeability would lead to oedema and an increase in tumour interstitial pressure further leading to vascular collapse. In addition, as the blood flow slows down, red cells stack up causing an increase in viscous resistance and slowing the blood flow by positive mechanisms. The consequence of this is the appearance of central necrosis with a viable peripheral rim of tumour cells, leading to rapid repopulation of tumours and the consequent failure to achieve significant tumour growth delay . The comIn conclusion, in this study, we provide evidence that electrochemotherapy, in addition to direct cytotoxic effect on tumour cells, has a vascular disrupting action. The vascular disrupting action is dual, namely the effect of application of electric pulses to the tumours that induce a rapid shutdown of tumour blood flow that recovers within 24 h and the continuation of the vascular disrupting effect of electrochemotherapy, predominantly through its cytotoxic effect on endothelial cells.


Thermosensitisation by step-down heating (SDH) has previously been demonstrated in experimental rodent tumours. The purpose of the study reported here was to investigate whether the SDH effect in tumours in part may be attributed to heat-induced alterations in the capillary network and/or the microenvironment. Two human melanoma xenograft lines differing substantially in vascular parameters were selected for the study. A thermostatically regulated water bath was used for heat treatment. The conditioning treatment (44.5 degrees C or 45.5 degrees C for 15 min) was given in vivo, whereas the test treatment  was given either in vitro or in vivo. Treatment response was measured in vitro using a cell clonogenicity assay. Fraction of occluded vessels following heat treatment was assessed by examination of histological sections from tumours whose vascular network was filled with a contrast agent. Tumour bioenergetic status and tumour pH were measured by 31P magnetic resonance spectroscopy. The conditioning heat treatments caused significant vessel occlusion, decreased tumour bioenergetic status and decreased tumour pH in both tumour lines. The SDH effect measured when the test treatment was given in vivo was significantly increased relative to that measured when the test treatment was given in vitro. The magnitude of the increase showed a close relationship to fraction of occluded vessels, tumour bioenergetic status and tumour pH measured 90 min after treatment with 44.5 degrees C or 45.5 degrees C for 15 min. The increased SDH effect in vivo was probably attributable to tumour cells that were heat sensitive owing to the induction of low nutritional status and pH during the conditioning treatment. Consequently, the SDH effect in some tumours may in part be due to heat-induced alterations in the microenvironment. This suggests that SDH may be exploited clinically to achieve increased cell inactivation in tumours relative to the surrounding normal tissues.


In 1998, a varicella-zoster virus glycoprotein E (gE) mutant virus (VZV-MSP) was isolated from a child with chickenpox. VZV-MSP, representing a second VZV serotype, was considered a rarity. We isolated another VZV-MSP-like virus from an elderly man with herpes zoster. These gE mutant viruses may have arisen through independent mutation or may represent a distinct VZV subpopulation that emerged more than 50 years ago. In 1998, a VZV mutant virus (VZV-MSP) was discovered in Minneapolis-St. Paul, Minnesota; the virus had a missense mutation in the preponderant surface glycoprotein called gE . The mutBefore 1995, a general assumption was that only one VZV serotype was found around the world ,11. WhetWe describe the second case of a gE mutant virus found in North America. This discovery provides evidence that the gE mutant virus was unlikely to have occurred as a serendipitous chance mutation.In December 1999, a 75-year-old man from Vancouver, British Columbia, Canada, arrived at the hospital with severe zoster lesions on his face . Painful postherpetic neuralgia with headaches, associated with occasional blurring of vision and lasting for 6 months, developed. The patient was treated with famciclovir (500 mg three times a day for 7 days). A swab of the facial vesicles was obtained from the patient during acute zoster before famciclovir treatment.Characteristic VZV cytopathogenic effect was seen after 9 days in cell culture. Results of immunofluorescence staining, performed with a commercial kit  by using the anti-gE monoclonal antibody (MAb 3B3), were negative. Staining with monoclonal antibody conjugates for cytomegalovirus and human herpes simplex viruses was also negative. Electron microscopy indicated the presence of enveloped virus characteristic of herpesviruses. Polymerase chain reaction amplification and subsequent DNA sequencing confirmed the virus genome to be VZV (hereafter referred to as VZV-BC). The VZV-BC strain was then analyzed in ORF68 (encodes gE and contains the 3B3 MAb epitope) and found to have an identical nucleotide sequence to VZV-MSP (GenBank accession no. AY005330). The VZV-BC and VZV-MSP strains differed from the prototype Dumas VZV strain (GenBank accession no. X04370) in ORF68 by a single G448A nucleotide mutation (D150N amino acid substitution) in the 3B3 epitope of gE . VZV-MSPVZV-MSP was the first community-acquired VZV strain shown to have a phenotype distinguishable from the traditional VZV phenotype. This case report documents the existence of a second isolate with the same gE genotype. VZV-MSP has been shown to have attributes consistent with increased virulence in the SCID-hu mouse model . Both VZThe origin of this variant may be the same gE mutation, occurring separately in two patients by chance. The argument favoring chance was raised after the discovery of the first gE mutant; however, we find this argument less compelling after the discovery of a second VZV strain with an identically mutated gE antigenic site, just 4 years later. Two other possibilities for its origin seem more plausible. The same gE mutation may have occurred from selective antibody pressure. In an article from the laboratory of the Nobel laureate Zinkernagel , the invWe conclude that diagnosticians should be aware of the existence of VZV-MSP-like strains and the potential for false-negative testing results with single Mab-based antigen-detection kits for VZV or other herpesviruses. Further clinical observations are needed to fully assess the biological significance of the VZV gE mutant virus.


Intrapleurally injected cells of an ascitic rat tumour produced intrapleural effusions and solid pleural deposits. BCG, or its methanol extraction residue (MER) injected into the pleural space, suppressed tumour development and prolonged survival. Treatment was effective if given a few days before or after tumour injection. In contrast, active specific immunotherapy by repeated s.c. injection of viable or radiation-attenuated tumour cells in admixture with BCG was unsuccessful, and did not improve the response to intrapleural BCG treatment.