Intratumour injection, commonly used for gene or drug delivery but also associated with needle biopsy or insertion of invasive measuring devices, may damage tumour microvessels. To examine this possibility, SCCVII tumours grown subcutaneously in C3H mice were injected with a 26 gauge needle containing 0.1 ml of the fluorescent dye Hoechst 33342 to label cells lining the track of the needle. Hoechst-labelled cells sorted from these tumours were more sensitive to killing by hypoxic cell cytotoxins  and less sensitive to damage by ionizing radiation. Hoechst-labelled cells also bound the hypoxia marker pimonidazole when given by i.p. injection. Intratumour injection transiently increased hypoxia from 18 to 70% in the tumour cells adjacent to the track of the needle. The half-time for return to pre-treatment oxygenation was about 30 min; oxygenation of tumour cells along the track had recovered by 20 h after intratumour injection. This effect could have significant implications for intratumour injection of drugs, cytokines or vectors that are affected by the oxygenation status of the tumour cells as well as potential effects on biodistribution via local microvasculature.British Journal of Cancer (2002) 86, 429–435. DOI: 10.1038/sj/bjc/6600059www.bjcancer.comThe Cancer Research Campaign© 2002  Intratumour injection is used to deliver a variety of agents, including drugs, genes and cytokines that are available in limited amounts or that do not normally localize to tumours .Therefore, experiments (reported here) were designed initially to measure the distribution of drugs given by intratumour injection. The bioreductive hypoxic cell cytotoxin tirapazamine was injected together with a rapidly bound fluorescent dye, Hoechst 33342, directly into SCCVII murine tumours. Hoechst 33342 binds to cells lining the track of the needle, but tirapazamine was expected to diffuse to hypoxic regions within the tumours. Interestingly, tumour cells most sensitive to tirapazamine were those cells that bound the most Hoechst 33342. This led us to question whether intratumour injection, and presumably other invasive procedures, may affect local tumour oxygenation.Our previous efforts to demonstrate an effect of intratumour needle puncture on average tumour oxygenation in experimental tumours were inconclusive  from a stock solution containing 20 mg ml−1 in sterile PBS. In one experiment, pimonidazole was injected i.t. at a dose of 2–50 mg kg−1. Ninety minutes after intraperitoneal injection of pimonidazole, corresponding to three plasma half-lives for this drug .Ethanol-fixed cells were centrifuged, washed once in PBS then resuspended in PST (PBS containing 4% serum and 0.1% Triton X-100). A FITC-conjugated (1 : 1000 dilution) primary antibody was added to 2×10vs logarithm of fluorescence anti-pimonidazole antibody intensity, were analyzed by a least-squares approach for three normal curves representing aerobic, intermediate and hypoxic tumour cell populations. The hypoxic fraction was defined as the percentage of the population that bound, on average, 10 times more pimonidazole than the aerobic population of cells  were prepared using a cryostat. Sections were examined immediately with UV excitation using a Zeiss axioplan epi-fluorescence microscope with CCD camera and image software. Areas that showed significant Hoechst 33342 fluorescence  were captured. Then sections were fixed in 70% ethanol (Hoechst 33342 is lost at this step) and stained for pimonidazole using the FITC-conjugated anti-pimonidazole monoclonal antibody (1 : 1000 dilution in PST). The same area of the tumour was re-examined using 488 nm excitation. Digitized images were pseudo-coloured and superimposed using Picture Publisher software.DNA single-strand breaks produced by tirapazamine were measured using the alkaline comet assay as previously described . DNA damvs 20.8±7.4, mean and standard deviation, n=6). Five minutes to 24 h later, tumours were excised, and a single cell suspension was prepared. Single cells were then analyzed by flow cytometry. Fluorescence within the fractions from an individual tumour varied 20–40-fold when tumour cells were assigned to 10 equal fractions. Fluorescence intensity increased dramatically in fractions 9 and 10, representing 10–20% of the tumour cells.Figure 1Intensity decreased slightly over the first 2 h following intratumour injection but was 4–5 times lower by 24 h after injection. Intravenous injection of 80 times more Hoechst 33342 (1.6 mg per mouse) produced a similar average intensity but a reduced maximum intensity (black circles) consistent with a larger percentage of tumour cells being exposed to the dye when delivered by intravenous injection.The distribution of DNA damage within SCCVII tumours after intratumour injection of tirapazamine together with Hoechst 33342 is shown in Figure 2Since the drug concentration was highest in cells along the track of the needle, one could argue that more damage occurred here simply because these cells were exposed to the highest drug dose. Therefore, response to ionizing radiation, which produces three times more killing in aerobic than hypoxic cells, was also used to determine whether cells bordering the needle track were hypoxic. Tumours that had been injected with Hoechst 5 min before exposure to radiation showed less killing of the brightly fluorescent cells compared to the dimmest cells  of Hoechst was injected  labelled a larger area of the tumour but were cleared at the same rate. This suggests that the slower recovery kinetics observed in this study is more likely to represent repair of damage to microvessels rather than clearance of the injected fluid. Jain and colleagues also noted that using a very slow infusion rate from a single point source, bulk flow was responsible for removal of fluid and reabsorption via blood vessels was minimal in the LS174T xenograft tumour (The effects we have observed here are confined to the immediate region of cells surrounding the sites of injection. This observation is consistent with previous studies that found only a small region of the RIF-1 tumour surrounding the site of D imaging . In thesThe typical hypoxic fraction in the SCCVII tumour of air-breathing mice is about 12% when measured using pimonidazole binding , arguingLocalized damage to the tumour blood vessels caused by invasive procedures is not unexpected. Previous reports have indicated that invasive techniques, such as fine needle aspiration biopsy, can occasionally produce extensive tissue damage (e.g. The fact that adjacent tumour cells are hypoxic and gradually reoxygenate has implications for the use of intratumour injection to deliver drugs. Biodistribution of agents delivered by this route is likely to be affected by damage to tumour microvessels. In addition, oxygen dependency for toxicity has been demonstrated for ionizing radiation, cytokines and several anticancer drugs (for a review, see


A tissue-isolated preparation of the P22 rat carcinosarcoma was used to investigate the tumour vascular response to angiotensin II (ATII). In particular, the relative importance of systemic and local tumour factors was assessed by comparing tumour vascular resistance during systemic administration of ATII and during administration directly into the tumour-supplying artery. The effect of hypervolaemia on tumour vascular resistance was determined as well as the effect of ATII on oxygen metabolism. Tumour vascular resistance was increased by ATII in a dose-dependent manner. The response was biphasic with an initial peak in resistance followed by a lower plateau phase. Systemic administration of ATII was more effective in increasing tumour vascular resistance than direct administration. This suggests that systemic administration is not causing any reopening of previously collapsed tumour blood vessels. Further evidence for this is that hypervolaemia caused no reduction in tumour vascular resistance and that there was no difference in oxygen extraction by tumours between groups treated with systemically and directly administered ATII. A heterogeneous distribution of ATII receptors in the P22 tumour is a more likely explanation for the known heterogeneity of blood flow response to ATII.


Numerous studies have suggested that digitalis derivatives promise to be superior to existing adjuvant therapy for breast cancer as to effects and side-effects. In the present study, we have used gene expression analysis to determine the molecular action of digitoxin on breast cancer cells and assessed digitoxin’s ability to synergize with the chemotherapy agent paclitaxel with respect to inhibition of cell proliferationWe treated  MDA-MB-453 human breast cancer cells with digitoxin at four doses {20 ng/ml (26 nM) to 1 μg/ml} and collected RNA at 6 h and 24 h for gene expression analysis. To examine the effects on ER positive cells, we treated MCF7 cells with digitoxin at 1 μg/ml and collected RNA for RT-PCR analysis. In addition, we assayed the growth inhibitory effect of low doses of digitoxin combined with paclitaxel and determined combination index values.+-K+-ATPase; digitoxin enhanced the growth inhibitory effects of paclitaxel on Her2 overexpressing breast cancer cells.To reveal primary effects, we examined digitoxin’s effect 6 h post-treatment with the highest dose, 1μg/ml, and found upregulation of the stress response genes EGR-1 and NAB2, lipid biosynthetic genes and the tumor suppressor gene p21, and downregulation of the mitotic cell cycle gene CDC16 and the replication gene PolR3B. RT-PCR analysis validated effects on stress response, apoptotic and cell cycle genes on MDA-MB-453 and MCF7 cells. Western blot analysis confirmed induction of EGR1 protein at 1 h and ATF3 at 24 h. Paclitaxel, as well as digitoxin, inhibited the in vitro activity of the purified NaOur studies show the potential of digitoxin to prevent and treat breast cancer and indicate that the combination of digitoxin and paclitaxel is a promising treatment for ER negative breast cancer. These findings are the first to alert physicians to the possible dangers to patients who take a combination of digitoxin and paclitaxel. The potential dangers ensuing when paclitaxel and digitoxin are combined are dependent on the dose of digitoxin. Numerous studies have supported the notion that digitalis derivatives promise to be superior to existing adjuvant therapy as to effects and side-effects.2Digitalis purpurea L. or common foxglove  has been used for centuries as a folk remedy for heart ailments. More than thirty cardiac glycosides have been isolated from foxglove leaves, including digitoxin and digoxin. These compounds possess a steroid nucleus with a characteristic 17-carbon skeleton, an unsaturated lactone ring at the C17 position, and one or more glycosidic residues at C3 as seen in +-K+-ATPase appears to be dependent on the specific cardiac glycoside compound as well as the subunit set of the receiving Na+-K+-ATPase which is related to the tissue type.There is evidence that cardiac glycosides also have antitumor activity. Breast cancers from women on digitalis have more benign characteristics evidenced by a 9.6 times decrease in the recurrence rate after five years following a mastectomy. However,Though it was initially thought that only toxic doses of digitoxin could be useful for anticancer activity, studies indicate that low doses of digitoxin induce apoptosis in malignant cell lines.6 Crude e+-K+-ATPase, a member of evolutionarily conserved enzymes that couple ATP hydrolysis to ion translocation across cellular membranes. was determined by the Taussky-Shorr method.μg/ml for 24 h and assayed the percent surviving cells. Cells that were pretreated with siRNA and then treated with digitoxin were also used to prepare extracts for Western blot analysis to confirm the ERK2 knockdown.To test the functional relevance of ERK2 (p42/44MAPK pathway), we examined the growth inhibitory effects of digitoxin on MDA-MB-453 cells using the model system RNAi-mediated gene knockdown. We pretrμg/ml of digitoxin for 6 and 24 h and MCF7 cells with digitoxin at 1 μg/ml for 6 and 24 h. Total cellular RNA was extracted using Trizol  according to the manufacture’s protocol with minor modifications, and then purified twice with Qiagen’s RNeasy column, as previously described. Total RNA (8 μg) was reverse transcribed with T7-(dT)24 primer and Super Script III reverse transcriptase (Invitrogen). After purification, cDNA was in vitro transcribed into biotin labeled antisense cRNA with the BioArray high yield RNA transcript labeling kit , according to a modified Affymetrix protocol. cRNA (15 μg) was fragmented into the final probe and hybridized to human U133A 2.0 gene chips , comprised of more than 22,000 probe sets.We treated MDA-MB-453 cells with 20 ng/ml, 0.1 (26 nM), 0.2 or 1 50, twice the IC50 or ten times the IC50 concentration, measured at 48 h, of digitoxin. Western blot analysis was performed as described previously.–26μg/ml,μg/ml, the three main genes that were significantly altered and possibly contribute to cancerous behavior were NAB2, ARID5B, and MCL1. SC5DL and EGR1 appear with P values slightly higher than 0.05, indicating their involvement as well. It appears that these are among the primary genes affected by digitoxin.In the cells treated with digitoxin at 0.2 μg/ml, for either 6 or 24 h. However, some of these genes were also activated after treatment with 0.1 or 0.2 μg/ml, for 6 or 24 h, including additional stress response genes . μg/ml for 6h, further repressed at 24 h, and slightly repressed after treatment with digitoxin at 0.2 μg/ml for 6 or 24 h. The cluster of genes displayed in μg/ml of digitoxin for 6 h, but not evident at 24 h. This cluster also contained lipid biosynthetic genes (INSIG1). A fourth region, expanded for CDKN1A, showed a progressive increase in expression after treatment with digitoxin at 0.1, 0.2 or 1 μg/ml for 6 or 24 h, with a more pronounced increase at 24 h (data not shown). This cluster also contained the stress gene DNAJB4 and the apoptotic gene GDF15.We used hierarchical clustering to reveal genes that are coordinately controlled . Figure P-values are indicated for all microarray and PCR genes in the Tables.To verify some of the digitoxin-induced changes that we observed in gene expression detected by microarray analysis, we performed real-time RT-PCR analysis on the same RNA samples. We examined 12 genes related to the stress response or cell cycle control. The RT-PCR results , b were μg/ml the expression of ATF3, ATF4 and DNAJB4 decreased from 6 to 24 h and the expression of HSF2 and PPP1R15A increased and then leveled off [μg/ml, they showed a progressive increase in mRNA expression; (3) expression of the apoptotic genes GDF15-A, CDKN1A and the Na+-K+-ATPase ATP1A1 progressively increased after treatment with digitoxin at all doses for 6 or 24 h [Consistent with the hierarchical clustering results, there were four main patterns of expression: (1) mRNAs for the ER stress gene EGR1 and the lipid genes INSIG1 and HMGCS1 increased at 6 h and decreased at 24 h ; (2) theeled off , whereas or 24 h , b; and  or 24 h . Thus, dμg/ml for 1 h, whereas the ATF3 protein was induced after treating with digitoxin at 1.0 μg/ml for 24 h [Digitoxin significantly upregulated the expression of the transcription factor ATF3 and the early response gene EGR1, by both microarray and RT-PCR analysis. Western blot analysis confirmed that when MDA-MB-453 cells were treated with digitoxin, EGR1 protein was induced after treating with digitoxin at 0.1, 0.2 or 1.0 for 24 h .μg/ml) for 24 h resulted in decreased cell proliferation (96.0% to 76.9%), indicating that MAPK1 is involved in the survival phase of the digitoxin-induced stress response [To confirm an effect on MAPK signaling, we examined the growth inhibitory effects of digitoxin using the model system RNAi-mediated gene knockdown. Pretreating cells with siRNA to MAPK1 (ERK2) before treating with digitoxin  the ER stress gene EGR1 and the lipid gene INSIG1 increased at 6 h and decreased at 24 h; 2) the stress genes ATF4, ATF3 and GADD34 showed a progressive increase in expression of the related mRNAs after treatment with digitoxin at 1 μg/ml for 6 or 24 h; and 3) the cell cycle gene CDC 16 showed a progressive decrease in expression [Figure To determine whether other breast cancer cell lines would react similarly to digitoxin, we tested the effect of digitoxin at 1 n Figure –c.in vitro activity of the purified Na+-K+-ATPase. The respective IC50 values for digitoxin and paclitaxel were 0.62 and 0.61 μM, respectively [To find an effective combination treatment, we tested the effects of paclitaxel, a common treatment for breast cancer, on the ectively . In viewμg/ml digitoxin and 0.5 nM of paclitaxel, and moderate synergy with 0.01 μg/ml digitoxin and 1 nM of paclitaxel. With the former combination , the percent viable cells decreased from 90.6% after treatment with paclitaxel alone to 42.9% after combination treatment, P<0.01 . Addition of digitoxin (0.01 μg/ml) to paclitaxel (1 nM) decreased cell survival from 63.2% to 30.3% (P< 0.01).Resulting data from combined increases of both paclitaxel (TAX) and digitoxin are shown in Ascertaining 1) the effectiveness of digitoxin at nontoxic doses, 2) its mechanism of action, and 3) its ability to synergize with chemopreventive and chemotherapy agents is essential to assess its chemopreventive/anticancer potential.Our studies indicated that low-dose digitoxin activated the expression of SRC-mediated signaling pathways, in particular genes induced by ERK1/2 including GRB7, RPS6KA5 and RAB15. High-dose digitoxin activated the expression of stress response and apoptotic genes, specifically, genes activated by the gene EGR-1 . These gμg/ml; since numerous factors can induce the expression of EGR1, it could be a sensitive target for cancer prevention or therapy. Our finding that EGR1 protein, a short-lived phosphoprotein, was induced after treating with digitoxin at 0.1, 0.2 or 1.0 μg/L for one h, and the ATF3 protein was induced after treating with digitoxin at 1.0 μg/ml for 24 h, contradicts the report of Perne et al.[As expected, digitoxin altered the expression of several genes involved in calcium homeostasis, including EGR1, IHPK2 and NR4A1 . EGR1, ane et al. that dig+-K+-ATPase activity in cardiac myocytes, which activates SRC and downstream ERK signaling cascades that eventually inhibit cell proliferation.[In some systems, EGR1 is thought to be downstream of the ERK pathway. Our resuferation.The effects of digitoxin on the expression of genes related to the ISR are not limited to the MDA-MB-453 cell line. Although the MCF7 cell line was less sensitive to the growth inhibitory effect of digitoxon, it exhibited increased expression of ATF4, DDIT3, GDF15, SLC7A11 and CYP1A1 in response to digitoxin treatment. The results of real-time RT-PCR analysis were remarkably similar between the two cell lines : digitoxμg/ml of digitoxin may be related to the ability of ERK to activate gene transcription mediated by sterols in HepG2 liver cancer cells.[Upregulation of lipid biosynthetic genes 6 h after treatment with 0.1, 0.2 or 1.0 er cells. This finμM). Since digitoxin inhibits the Na+-K+-ATPase, we reasoned that other inhibitors of ERK1 might inhibit the ATPase. We tested the effect of paclitaxel, which is frequently used to treat breast cancer, on the in vitro activity of the purified Na+-K+-ATPase and found it was a potent inhibitor. Consistent with our findings, paclitaxel has been shown to competitively inhibit ATP binding activity of the NTPase/helicase of hepatitis C virus with an IC50 of about 22 μM,[+-K+-ATPase.The optimal treatment for breast cancer most likely requires a combination of agents or modalities. Digitoxin’s upregulation of ERK resembled the effects of paclitaxel (TAX) in human ovarian SKOV3 cells. At low cut 22 μM, suggestiPaclitaxel is known to arrest cells in the M phase of the cell cycle, whereas About one-third of invasive breast cancers are ER-negative. It is important to develop novel therapeutic agents which have an acceptable toxicity profile and activity against ER-negative breast cancer. We therefore investigated the growth inhibitory effect of combinations of digitoxin with paclitaxel on Her2 overexpressing, ER low MDA-MB-453 human breast cancer cells and found moderate synergy. The ability of digitoxin to potentiate paclitaxel’s effects at low concentrations on ER-negative human breast cancer cells may permit the use of lower doses of this toxic chemotherapy agent and could delay the development of resistance to paclitaxel in cancer treatment. ER negative, Her2 overexpressing patients have a poorer clinical prognosis, do not benefit from adjuvant hormonal therapy, and are at risk for recurrence and second breast primaries, giving this study more clinical relevance.It is vital to the future practice of cancer medicine to develop a therapeutic agent that has an acceptable toxicity profile yet is active against estrogen receptor (ER)-negative breast cancer. This is one of the most aggressive cancers and afflicts about a third of the victims of the disease. Furthermore, the optimal treatment for most breast cancers requires a combination of agents. Our work addresses both these requirements.Our studies indicate the ability of digitoxin to activate transcription of apoptotic factors, repress cell cycle related genes and, at low concentrations, enhance growth inhibition when combined with paclitaxel. Our findings thus suggest that digitoxin, alone and when combined with paclitaxel, is a promising treatment for ER-negative breast cancer. In addition, our findings are the first to alert physicians to the possible dangers to patients who take a combination of digitoxin and paclitaxel. The potential dangers ensuing when paclitaxel and digitoxin are combined are dependent on the dose of digitoxin.


Amniotic fluid Gram stain and culture have been utilized as laboratory tests of microbial             invasion of the amniotic cavity. The Gram stain of amniotic fluid has a low sensitivity in the             detection of clinical infection or microbial invasion of the amniotic cavity, and amniotic fluid             culture results are not immediately available for management decisions. Glucose             concentration is used to diagnose infection in other sites such as cerebrospinal fluid.        Objective: The purpose of this study was to evaluate the usefulness             of amniotic fluid glucose concentration in detecting microbial invasion of the amniotic             cavity associated with preterm labor and preterm premature rupture of membranes.                    Methods: Amniocentesis was performed in 60 women with preterm labor             and/or preterm premature rupture of membranes. Gram stain and culture for Mycoplasma hominis,             Ureaplasma urealyticum, aerobic, and anaerobic bacteria were performed. Subjects             were studied prospectively for the development of positive amniotic fluid cultures and the             development of clinical chorioamnionitis.                    Results: The diagnosis of clinical chorioamnionitis was made in             25% (15/60) of women entered into the study. Low amniotic fluid glucose concentration            Was considered < 15 mg/dl. The sensitivity, specificity, and positive predictive value             of low amniotic, fluid glucose concentration to predict clinical chorioamnionitis were 73.3%,             88.1%, and 68.8% respectively, while positive amniotic fluid culture, hada sensitivity of             43.8%, specificity of 79.5%, and positive predictive value of 43.8%.                    Conclusions: Amniotic fluid glucose concentration was more sensitive             in predicting chorioamnionitis than either Gram stain or culture. Amniotic fluid glucose             concentration was better in predicting clinical chorioamnionitis than predicting positive             amniotic fluid culture results. Gestational age-dependent normal ranges and pathologic             conditions that may alter amniotic fluid glucose concentrations should be considered             when interpreting amniotic fluid glucose values to diagnose microbial invasion of the             amniotic cavity.


Pre-treatment with the anabolic steroid nandrolone decanoate (ND) increases the LD50 of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 5-Fluorouracil (FU) in NMRI mice. Administration of ND did not affect the anti-tumour action of CCNU against a transplantable mouse adenocarcinoma of the colon (MAC 13) or the anti-tumour action of FU against MAC 26. In both tumour lines ND had no significant effect on tumour growth. These data suggest that an increase in the anti-tumour selectivity of these agents may be produced by pre-treatment with ND.


Renal tumours were induced in dietary-primed rats by injection of dimethylnitrosamine. Control and tumour tissue was excised at varying periods and maintained in short-term organ culture in the presence of 3H- or 14C-fucose. The plasma membranes were then isolated, and the isotopic profiles of normal kidney and renal tumour membrane proteins were established, using polyacrylamide-gel electrophoresis in dodecyl sulphate. Several fucose-containing glycoproteins of the plasma membranes were found to alter upon neoplastic transformation: 4 increased and 3 decreased. The probable identity of 2 of these proteins is indicated: alpha-foetoprotein is one of the glycoproteins which increased, whereas neutral endopeptidase decreased in the tumour membranes. Fluorescein-labelled lectin binding by the kidney tissue was also found to alter upon transformation. The most marked changes were an increase in sialic acid (neuraminidase-sensitive) and galactosamine (Ricinus communis agglutinin Type I) in the nuclei of some neoplastic cells and some hyperplastic-tubule cells.


Various doses (1 microgram to 10 mg) of oestriol (E3) were intraperitoneally injected into mice immediately after subcutaneous inoculation of an oestrogen receptor-negative lymphoma cell line (KE-5) established from a spontaneously developed AKR thymic lymphoma. The growth of KE-5 cells was markedly promoted by E3 at the early stage of tumour growth. At this stage, 1 microgram E3 enhanced tumour growth significantly and the maximum effect was obtained with 1 mg E3. Normal female mice showed a higher incidence and shorter latency than males. However, once tumours became palpable, the tumour growth rate appeared to be unaffected. Histological observations using Alcian blue and colloidal iron revealed a marked increase of hyaluronic acid in the subcutaneous connective tissue of the tumour-injection site within 3-5 days after intraperitoneal administration of 1 mg E3. Biochemical analyses showed a rapid and marked increase in skin hyaluronic acid content to over 3 times the control levels (0.25 +/- 0.10 mg g-1 skin) within 3 days of E3 administration. Subcutaneous inoculation of KE-5 cells together with hyaluronic acid (0.2 mg) resulted in markedly enhanced tumour growth, particularly at the early stage. These results suggest that an increase in stromal hyaluronic acid content is the most likely mechanism responsible for the promoting effect of E3 on KE-5 cells.


Hypothyroidism, alone or combined with progesterone, significantly decreased 7,12-dimethylbenz(a)anthracene (DMBA) mammary tumorigenesis relative to controls. However, the decrease was less in the progesterone-treated group, and statistical analysis showed that progesterone enhanced tumorigenesis to the same extent in hypothyroid animals as in the controls. Most tumours in hypothyroid progesterone-treated rats were adenocarcinomata; in the absence of the hormone most tumours were benign. However, the difference between the tumour types in the 2 groups was not statistically significant.The morphological changes observed in the endocrine glands, genital tracts and non-neoplastic mammary tissue, considered in relation to previously reported data, suggest that hypothyroidism reduced the tumour yield mainly by secondarily inhibiting somatotrophin production and secretion, although the effect of decreased food intake could not be excluded completely. The higher tumour yield in the hypothyroid progesterone-treated rats may have been due to higher circulating levels of prolactin in this group compared with those in the hypothyroid group which received no hormone.


Introduction. Dermatofibrosarcoma Protuberans (DFSP) is a rare cutaneous tumour of low/intermediate malignant potential, which occasionally arises on the vulva. Historically, the treatment has been wide local excision (WLE). Mohs micrographic surgery (MMS) is now recommended to ensure precise margin control. MMS to treat DFSP of the trunk and extremities is well documented. However, no report to date has described its use in vulval DFSP. Case History. A 39 year old woman presented with a longstanding nodule in the left labium majus. Histology after surgical removal showed an incompletely excised DFSP. MMS was undertaken with primary closure of the defect. Three years following treatment there is no evidence of recurrence. Discussion. The local recurrence rate of DFSP after WLE ranges from 0–75%. Finger-like projections from DFSP into surrounding tissue often results in incomplete excision. Representative vertical sections used in WLE assess less than 1% of the total tumour margin. MMS uses systematic horizontal sectioning. 100% of the tumour margin is microscopically examined. MMS is now advocated to ensure precise margin control. Dermatofibrosarcoma protuberans (DFSP) is a rare cutaneous tumour of low/intermediate malignant potential, which occasionally arises in the vulva. Historically, the treatment has been wide local excision (WLE). However, local recurrence rates after WLE are high . MicroscMohs Micrographic Surgery (MMS) offers an alternative therapeutic approach. MMS allows mapping of the extend of DFSP with microscopic examination of its deep and lateral margins. 100% of the tumour margin is examined. The microscopic tumour projections into surrounding tissue are reliably identified and excised. Favourable outcomes are reported with preservation of tissue and a low recurrence rate [MMS to treat DFSP of the trunk and extremities is well documented . HoweverA-39-year old woman presented with a longstanding painless nodule in the left labium majus. Histology after surgical removal showed an incompletely excised DFSP. Chest X-Ray showed no evidence of metastasis. Further management involved a multidisciplinary team, consisting of a dermatologist, gynaecological oncologist, and histopathologist. A residual 10–15 mm nodule was first debulked under local anaesthetic. MMS was then undertaken. A highly specialized and accurate protocol of surgical technique were followed. Anatomic orientation of the specimen was defined and drawn onto a map. The specimen was then sliced so that deep and lateral margins lay in the same plane. Horizontal frozen sections were stained with haematoxylin and eosin and examined microscopically to provide a complete 360 degree view of the tumour margins. With MMS the microscopic projections into surrounding tissue can be identified. Residual tumour is then depicted onto the previously drawn anatomic map to guide excision. A further tissue layer is removed and the same meticulous procedure is followed until microscopic examination shows no residual tumour. In our patient the tumour was completely removed after one stage of MMS. The defect was repaired with primary closure. Three years following treatment, there is no evidence of recurrence.DFSP is a rare neoplasm accounting for 0.1% of all malignancies . It typiDFSP of the clitoris and vulva are rare. DFSP of the vulva typically arises in the left labium majus . The reaThe tumour often presents as a solid, protuberant nodule. Due to its indolent nature, it often escapes detection in the early stages. Pain and ulceration may occur with tumours in an accelerated growth phase . Aggressive local growth is characteristic. MRI can identify the extent of the tumour and its relation to adjacent structures . Lymphatic spread of DFSP is rare . MetastaThe tumour most probably arises from a stem cell within the dermis. Local growth exhibits fingerlike projections into surrounding tissue. Subsequently, it can invade the subcutaneous tissue, underlying fasciae, muscle and bone . Correct histological diagnosis can be problematic as it is often difficult to distinguish DFSP from other fibrohistiocytic neoplasms . ComprehHistology typically shows elongated fibroblasts with small nuclei arranged in a “storiform” pattern. Compared to fibrous histiocytomas DFSP displays a more uniform appearance with little nuclear pleomorphism and low to moderate mitotic activity. Absence of necrosis and immunohistochemical staining for CD34 differentiate DFSP from fibrous histiocytomas [Historically, the treatment for DFSP has been wide local excision (WLE) of both primary and recurrent lesions.The local recurrence rate of DFSP after WLE ranges from 0%–21% for trunk sites and 50%–75% for head and neck sites [MMS uses systematic horizontal sectioning with 100% of the tumour margin being microscopically examined. This increases the likelihood of any tumour projections being detected.Since MMS allows for accurate margin control, maximum preservation of healthy tissue with complete excision can be achieved. It, therefore, leaves the smallest post surgical defect and minimises recurrence. Nearly 75% of such wounds can be managed with primary closure. Tissue conservation was important in our case to preserve function, with the DFSP being close to the clitoris. Wide local excision with 3 cm margins as previously recommended  may haveMMS is now advocated to ensure precise margin control. The favorable success rate of MMS compared to WLE in patients with DFSP is well documented . Site diThe local recurrence rate after the first MMS treatment for all patients with DFSP, has been reported as 7% over a 5-year period. 5 year cure rates have been reported as 92% for head and neck sites, 94% for trunk sites and 100% for DFSP in extremity sites. The 5-year cure rate for all sites was 93% after the first MMS treatment and 98.5% after the second [Local recurrence after MMS may be related to “skip” areas of tumour that become separated from the main tumour mass. However 25% of local-recurrences develop after 5 years . Long teWLE for DFSP of the vulva has previously been described. MMS for DFSP of the vulva represents, however, a novel treatment. Treatment outcomes for DFSP at other sites are uniformly more favorable with MMS when compared with standard WLE . This caWe, therefore, propose that MMS is considered in all cases of primary or recurrent DFSP of the vulva.


Whereas tumour-infiltrating lymphocytes were nonresponsive in the proliferation assays, lymphocytes from sentinel nodes proliferated dose dependently and secreted interferon-γ upon stimulation with tumour homogenate. The responses were of varying magnitude and tended to be weaker in metastatic sentinel nodes. Sentinel node lymphocytes represents an enriched source of tumour reactive lymphocytes, and may be useful in future trials of adoptive immunotherapy.The sentinel node is the first lymph node to receive lymphatic drainage from a tumour and is usually the first site of metastases. Today, the sentinel node is used for tumour staging. Here, we focus on its immunological role and investigate lymphocytic function in sentinel nodes, identified intraoperatively by peritumoural dye injection, from 15 patients with colon cancer. Tumour infiltrating lymphocytes, sentinel and nonsentinel lymph node cells and peripheral blood leukocytes were studied by flow cytometry, proliferation assays and interferon- Colorectal cancer (CRC) is a major cause of morbidity worldwide, accounting for over 1 million new cases and 500 000 deaths per year . StandarStudies of prognostic factors in colorectal cancer point out the histological status of the regional lymph nodes as the most important predictor of survival , is estain vitro in IL-2, display specific cytokine secretion in response to autologous tumour cells  was injected superficially in the serosa around the tumour. Within 5 min, one to four blue-coloured mesenteric lymph nodes were identified macroscopically as sentinel nodes and they were marked with sutures.The sentinel and nonsentinel nodes were cut in half. Slices <1 mm thick were cut from the central and the peripheral part of the nodes for flow cytometry and proliferation analysis. The rest of the node underwent routine histopathological examination. Tumours were histopathologically classified as Duke's stages A–D  Table 1Table 1. 6 cells sample−1 were subjected to investigation using flow cytometry (FACS). Cells were washed in PBS containing 2% FCS and 0.05% NaN3 and stained with fluorophore conjugated antibodies against the cell surface molecules CD4 PE, CD8 PerCp  and the very early activation marker CD69 FITC . After staining, cells were investigated using a FACSCalibur (Becton Dickinson) and data were analysed using the Cellquest computer software (Becton Dickinson).Peripheral blood leukocytes (PBL), lymph node cells and tumour cell suspensions at 1 × 10−1) of 2 × PBS followed by 5 min denaturation at 97°C. Tumour homogenates were diluted to 1 : 10 and 1 : 100 in complete proliferation media. Purified PBL and lymph node cells were used at 3 × 105 cells well−1 in time course proliferation assays against diluted tumour homogenate, Con A 10 μg ml−1 (Sigma) or carcinoembryonic antigen 100 μg ml−1 (Sigma) in triplicate. Proliferation was measured by adding 1 μCi of 3H-Thymidine/well (Amersham) 18 h prior to harvesting. Samples were subjected to scintillation counting.Single cell suspensions from lymph nodes and tumours were obtained by gentle pressure using a loose fit glass homogeniser. Peripheral blood leukocytes were purified by ficoll-paque . Cells were resuspended and washed twice in RPMI 1640  containing 2.5% fetal calf serum (FCS) (Life technologies). Finally, cells were resuspended in RPMI 1640 proliferation media containing 10% human AB serum , 1% penicillin–streptomycin (Sigma) and 1% glutamine (Sigma). Tumour samples were homogenised using a Ultra-turrax in 5 v . The amount of secreted IFN-γ was measured with ELISA  on culture supernatants in pooled samples of the triplicates.Stimulations for the measurement of IFN-One to four sentinel nodes (average 2.3) were detected intraoperatively using Patent blue injection in the circumference of the tumour. Patient characteristics and location of the tumours are presented in + and CD8+ cells in the lymphocytic gate varied between lymph nodes (+ to CD8+ cells and tumour stage or proliferative response (data not shown). Similar numbers of activated CD4+CD69+ lymphocytes were found both in sentinel and nonsentinel nodes regardless of absence .Single cell suspensions of lymphocytes collected from the tumour, sentinel and nonsentinel lymph node specimens, and peripheral blood (PBL), were prepared as described in Material and Methods. The number of sentinel nodes obtained and average cell yield from the sentinel node specimens for each patient are given in ph nodes . No sign3H-Thymidine incorporation assays using homogenised denatured autologous tumour cell extracts as antigen sources (μg ml−1 of CEA did not result in proliferation (data not shown). In nonsentinel nodes proliferation upon stimulation with tumour extract was not detected, with the exception of three patients  (The proliferative responses of the cell populations were characterised by time course  sources . TILs di sources  with an  sources . In addi7 and 8) . In PBLs7 and 8) . They reTo further investigate the functional state of the lymphocytes, T cells were stimulated with Con A which by-pass the antigen-specific activation through the T-cell receptor. TILs were unresponsive to Con A stimulation, as were SN lymphocytes from Duke's C patients no 11–12. SN lymphocytes from the remaining patients proliferated vigorously, as did PBLs and nonsentinel node lymphocytes from both C and D patients .γ when stimulated with antigen or with Con A was investigated in six patients, no 4, 5, 8, 13–15 (γ when stimulated with Con A (717 pg ml−1) and five times the background level upon addition of tumour extract, in spite of their inability to respond in the proliferation assay. Tumour-infiltrating lymphocytes from patient 5 (Duke's B) . In patients 8, 13 and 14 (Duke's C), the amount of TILs obtained, did not suffice for the IFN-γ ELISA.The ability of the lymphocytes to produce IFN-8, 13–15 . Tumour-uke's B)  and patiγ 4–38 times above background level when stimulated with autologous tumour homogenate. In patients no 14 and 15 SN lymphocytes did not produce IFN-γ above background, upon stimulation with tumour homogenate. Lymphocytes from nonsentinel nodes did not respond with any IFN-γ secretion above background levels  minor responses and an overall increase in survival was observed (In a murine system, tumour sensitised but not fully functional, lymphocytes were identified in tumour draining lymph nodes. These cells could be converted into effector cells by in vivo expanded source of tumour reactive lymphocytes. Instead of using TIL or peripheral blood lymphocytes, we propose sentinel node-derived lymphocytes as a source of enriched tumour reactive T cells, suitable for adoptive immunotherapy.In summary, we demonstrate that freshly isolated lymphocytes from sentinel nodes in patients with established colorectal carcinoma mount proliferative responses against autologous tumour extract. These cells have not yet been exposed to the immunosuppressive milieu of the tumour and constitute an


The present study examined the ability of human monocytes to produce reactive oxygen intermediates after a contact with tumour cells. Monocytes generated oxygen radicals, as measured by luminol-enhanced chemiluminescence and superoxide anion production, after stimulation with the tumour, but not with untransformed, cells. The use of specific oxygen radical scavengers and inhibitors, superoxide dismutase, catalase, dimethyl sulphoxide and deferoxamine as well as the myeloperoxidase inhibitor 4-aminobenzoic acid hydrazide, indicated that chemiluminescence was dependent on the production of superoxide anion and hydroxyl radical and the presence of myeloperoxidase. The tumour cell-induced chemiluminescent response of monocytes showed different kinetics from that seen after activation of monocytes with phorbol ester. These results indicate that human monocytes can be directly stimulated by tumour cells for reactive oxygen intermediate production. Spontaneous monocyte-mediated cytotoxicity towards cancer cells was inhibited by superoxide dismutase, catalase, deferoxamine and hydrazide, implicating the role of superoxide anion, hydrogen peroxide, hydroxyl radical and hypohalite. We wish to suggest that so-called ‘spontaneous’ tumoricidal capacity of freshly isolated human monocytes may in fact be an inducible event associated with generation of reactive oxygen intermediates and perhaps other toxic mediators, resulting from a contact of monocytes with tumour cells. © 1999 Cancer Research Campaign


Calculations were made to test whether the hypothesis of a multistep dissemination of the tumour from the primary site was likely. A multistep dissemination means that the presence of metastases at peripheral sites is influenced by the presence of the tumour in 3 organs, Several interpretations are discussed, with emphasis on those findings which could be explained in terms of a haematic route of dissemination or in terms of an anatomical proximity.


We hypothesised that Imatinib, a specific PDGFR-β inhibitor will, in addition to p-PDGFR-β inhibition, downregulate VEGF, decrease IFP and improve tumour oxygenation. A549 human lung adenocarcinoma xenografts overexpressing PDGFR-β were grown in nude mice. Tumour-bearing animals were randomised to control and treatment groups (Imatinib 50 mg kg−1 via gavage for 4 days). Interstitial fluid pressure was measured in both groups before and after treatment. EF5, a hypoxia marker, was administered 3 h before being killed. Tumours were sectioned and stained for p-PDGFR-β, VEGF and EF5 binding. Stained sections were viewed with a fluorescence microscope and image analysis was performed. Imatinib treatment resulted in significant reduction of p-PDGFR-β, VEGF and IFP. Tumour oxygenation was also significantly improved. This study shows that p-PDGFR-β-overexpressing tumours can be effectively treated with Imatinib to decrease tumour IFP. Importantly, this is the first study demonstrating that Imatinib treatment improves tumour oxygenation and downregulates tumour VEGF expression.Elevated intratumoral interstitial fluid pressure (IFP) and tumour hypoxia are independent predictive factors for poor survival and poor treatment response in cancer patients. However, the relationship between IFP and tumour hypoxia has not yet been clearly established. Preclinical studies have shown that lowering IFP improves treatment response to cytotoxic therapy. Interstitial fluid pressure can be reduced by inhibition of phosphorylated platelet-derived growth factor receptor- Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers and is the leading cause of cancer-related deaths worldwide   suspended in 200 μl of HBSS were injected subcutaneously into the right hind limb of each mouse.Adult female nude mice were used as host animals for xenograft tumours. Tumours were grown from exponentially growing A549 cell cultures. The A549 cell line was derived from human lung carcinoma and was purchased from ATCC. Cells were grown in RPMI 1640 media supplemented with 5% bovine calf serum. Cells (∼5.0 × 103. Tumour volume (V) was determined by the following equation: V=(L × W2) × 0.5, where L is the length and W is the width of the tumour. Tumour-bearing animals were then randomised into control  and treatment groups. There were 10 animals in each treatment group. Imatinib , 50 mg kg−1, was administered to tumour-bearing mice once a day for 4 days. The study was repeated twice. Imatinib was commercially obtained in a 100 mg tablet form, crushed and re-suspended in saline and given via gavage.Tumour growth was monitored twice weekly until they reached an average volume of 100 mmAnimal experiments were approved by the Institutional Committee on Research Animal Care and were performed in strict accordance to the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing and Education  and in accordance with the UKCCCR guidelines .−1). Interstitial fluid pressure measurements were performed for each animal before and 3 h after the last saline or Imatinib treatment. Tumour IFP was measured using the wick-in-needle technique as described previously . This set-up enabled continuous and stable recording of fluid pressure. Fluid communication between the needle and the transducer was confirmed by compression and decompression of the tubing during each measurement. Tumour IFP was determined by calculating the mean of two readings. Interstitial fluid pressure values in muscle and subcutaneous tissue served as controls.Animals were anaesthetised using nembutal (75 mg kgM EF5) of the hypoxia marker EF5 (2-(2-nitro-1H-imidazole-1-yl)-N- acetamide), generously provided by Dr Cameron Koch, University of Pennsylvania, 3 h before tumour excision. After killing the animals, the tumour specimens were removed and snap frozen in liquid nitrogen. Specimens were stored at −80°C until sectioned.Mice received an intravenous (i.v.) injection . Serial tissue sections were placed onto poly-L-lysine-coated slides . Consecutive tissue sections were cut at the largest circumference of the tumour. The sections were fixed in 4% paraformaldehyde in PBS (pH 7.2) for 1 h and incubated in the presence of primary antibodies. Primary antibody signals were visualised and enhanced by fluorescence-conjugated secondary antibodies to obtain sufficient fluorescence signal for imaging. Sections were rinsed three times for 2 min in PBS between each consecutive step of the staining procedure. Unless stated otherwise, all antibodies were diluted in PBS with 0.1% bovine serum albumin and 0.1% Tween-20 (PBS-BT). Omission of the primary antibody served as a negative control.Frozen specimens were cut into 10–12 β  diluted 1 : 400. The sections were then incubated for 1 h at room temperature with Texas Red-conjugated donkey anti-goat 1 : 200 .Tissue sections were incubated overnight at 4°C with goat polyclonal antibody to p-PDGFR-μl ml−1 .Tumour hypoxia was assessed immunohistochemically. EF5, a 2-nitroimidazole, is selectively retained in hypoxic tissues because it is metabolised to a free radical form in cells that binds to proteins to form specific residues in tissue that can be recognised by antibodies  diluted 1 : 200 and then incubated for 1 h at room temperature with FITC-conjugated goat anti-rat antibody 1 : 100.Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 15 min. Tumour sections were then blocked with 10% donkey serum for an additional 15 min. Sections were incubated overnight at 4°C with rabbit polyclonal antibody against VEGF diluted 1 : 100 . After rinsing 3 × 5 min with PBS, biotinylated donkey anti-rabbit antibody  was applied for 30 min at room temperature. The slides were washed with PBS for 3 × 5 min, followed by application of an avidin–biotin complex . Location of the reaction was visualised with 3, 3′-diaminobenzidine tetrahydrochloride .The tumour sections were quantitatively analysed with a semiautomatic method based on a computerised digital image analysis system. A high-resolution intensified solid-state camera on a fluorescence microscope  with a computer-controlled motorised stepping stage was used. Each tumour cross-section was sequentially scanned two times at × 50 magnification, using different filters for the FITC (green) and Texas Red and Cy3 (red) signals.After each scan, a high-resolution composite digital image was reconstructed from the individual microscopic fields. The whole scanning procedure thus yielded two composite images from each tumour section. The thresholds for positive staining were set to be background staining for each individual marker.β) were segmented using a single threshold value for all slides and all areas >2 pixels (72 μm2) in size were considered as positive regions. The stained fraction (%) for each marker was then calculated by dividing the stained area by the total area to give the hypoxic fraction and the relative p-PDGFR-β expression. To assess mean vessel density (MVD), the number of vessels, determined by positive CD31 staining, were counted in 6–8 fields and averaged to get a mean value for each tumour. The representative images for publication have been adjusted for brightness, contrast and colour balance for ease of viewing, but quantification was performed without any manipulation of the above-mentioned parameters.Haematoxylin and eosin staining on previously immunostained sections permitted contour lines to be drawn around the tumour area and exclusion of necrotic areas that were excluded from the quantitative analysis of each marker. The positively stained regions  for control samples and 13±2.2% (mean±s.e.m.) for the Imatinib-treated group. This decrease in p-PDGFR-β expression after Imatinib treatment was statistically significant (P=0.001)  .β affects tumour angiogenesis, immunohistochemisty studies were performed to determine VEGF and CD31 expression (P=0.004) (P=0.05) (To determine whether downregulation of p-PDGFR-pression . VEGF waP=0.004) . Mean ve(P=0.05) .vs the control group. Interstitial fluid pressure measurements were taken before and after 4 days of treatment. In controls, IFP averaged 1.4 and 2.1 mmHg before and after sham treatment, respectively. This difference was not significant (P=0.27). The Imatinib group exhibited similar pre-treatment values (1.53±0.23 mmHg). However, after 4 days of Imatinib treatment, IFP was reduced to 1.09±0.2 mmHg. This difference was statistically significant (P=0.036)  .P=0.008)  .β, EF5, VEGF and MVD were examined after 4 days of Imatinib treatment. Interstitial fluid pressure was positively correlated with EF5 staining   . This fiβ and VEGF expression. These changes in turn affected angiogenesis (i.e. MVD), IFP and oxygenation. The increase in tumour oxygenation following Imatinib treatment was most likely due to a combination of these effects.To our knowledge, this is the first study to demonstrate that the use of Imatinib in NSCLC xenografts results in decreased p-PDGFR-Aberrant tumour angiogenesis results in tortuous, leaky and nonfunctional tumour vasculature. The aberrancies in vascular structure and function play a crucial role in tumour growth and metastasis. For instance, therapeutic drug delivery is impaired thereby reducing treatment effectiveness and patient prognosis .β plays a crucial role in smooth muscle cell and pericyte recruitment. In normal angiogenic processes, interaction of these cells with ECs consequently results in stabilisation of the capillary wall  will decrease the percentage of tumour vessels with pericyte coverage, induce EC apoptosis and tumour regression (β and VEGF inhibition (Vascular smooth muscle cells and pericytes are essential for early growth of neovessels . Phosphoβ inhibition and VEGF downregulation. Whether or not this effect occurs preferentially on more immature blood vessels cannot be determined from this work because CD31, the EC marker used in this study, stains both mature and immature ECs.Data from this study shows that Imatinib reduces tumour angiogenesis, presumably through simultaneous p-PDGFR-β inhibition using Imatinib (Increased IFP within a tumour is a result of the tumour's highly permeable microvascular network and the absence of a functioning lymphatic system. Solid stress, generated by tumour cells growing in a confined space, also contributes to elevated IFP . The losImatinib .Tumour hypoxia is an independent predictive factor for distant metastasis and poor treatment outcome. Hypoxia can lead to ionising radiation and chemotherapy resistance by depriving tumour cells of the oxygen essential for the cytotoxic activities of these agents . In addiβ inhibition and decrease of IFP. A clinical trial investigating the role of Imatinib as an adjunct to chemotherapy in patients with metastatic, PDGFR-β-positive, NSCLC is ongoing. The working hypothesis of the study is that blockade of PDGFR-β will change IFP and subsequently effect tumour perfusion/permeability, drug delivery and treatment response. Further, by improving tumour oxygenation, we hope to enhance the cytotoxic and radiation therapeutic effect in cancer patients, improve patients' treatment response and increase overall survival. Further preclinical studies are in place to evaluate a potentially pertinent clinical role for a p-PDGFR-β inhibitor as a radiation sensitiser (We argue that drug delivery could be improved by p-PDGFR-nsitiser .


The absence of discernible abnormal symptoms such as pain, often leading to delayed diagnosis in cancer patients, may be indicative of a dysregulation in sensory transmission between the tumour and the central nervous system. We explored expression of Fos protein in spinal cord neurons in rats, during the development of the MAT-LyLu prostatic adenocarcinoma grafted on the hind limb. The tumour triggered the densest Fos labelling in the L3–L5 lumbar segments, ipsilateral to the grafted limb. The labelling, detected at day 5, increased until day 10 and dropped off thereafter. The ventral horn (except lamina IX) was the most densely labelled region. Histological examination of the grafted limbs demonstrated that no inflammatory reaction accompanied the tumour growth. Rats exhibited no behavioural alterations either spontaneous or induced by handling. These results demonstrate that signals are sent to the central nervous system by the peripheral tumour. Considering both the behavioural and histological observations, it is unlikely that spinal activity reflects a painful state. The nature of these signals, inefficient to trigger the appropriate reaction of the organism against the tumour, remain to be determined with regard to the pharmacologically active compounds synthesized and released by the tumour cells. © 1999 Cancer Research Campaign


We appreciate Dr Grant's comments  on our dDr Grant focused his comments on 2 aspects of our manuscript. First, he reiterates the similarities between our findings and those by others  but critSecond, Dr Grant notes that we failed to suggest a mechanism for the link. As indicated in our discussion , the pre


The results of 24 h food preference tests have suggested that learned food aversions may be involved in the development of anorexia in tumour bearing rats and in patients with cancer. We have performed similar tests over longer periods, up to 10 days, in male rats implanted with Leydig cell tumours, using semisynthetic diets containing differing proportions of fat, protein and carbohydrate. Tumour growth caused anorexia (16-30% decrease in food intake) and cachexia . Both tumour bearing and control rats preferred a high carbohydrate diet to a high fat diet regardless of their previous diet: tumour bearing rats showed no evidence of a learned food aversion in these experiments. Tumour bearing rats did show an initial preference for a novel high protein diet when this was offered as an alternative to the normal protein diet they had previously been consuming, but this apparent learned food aversion disappeared on the second day of the test and was in fact reversed on all the subsequent days of the test. However, tumour bearing rats did show a sustained preference for a novel low protein diet when this was offered as an alternative to the normal protein diet they had previously been consuming. These results suggest that anorexia in the tumour bearing rats was not caused by a learned food aversion. However the results do indicate that the tumour bearing rats may have developed a specific aversion to protein in the diet. Leydig cell tumours are known to secrete large amounts of oestradiol. However injections of oestradiol in normal male rats caused an increase in body fat content and had no effect on the rats' preference for dietary protein. Clearly hypersecretion of oestradiol was not responsible for the loss of body fat, the fluid retention and the aversion to dietary protein which characterised the tumour bearing rats. The mechanisms by which tumour growth causes anorexia and cachexia in these rats remains obscure.


I.p. immunization with gamma-irradiated hepatoma cells induces resistance to s.c. tumour-cell challenge in syngeneic WAB/Not rats. Mild heat treatment of these cells (greater than 41 degrees C for 30 min) destroyed this immunoprotective effect, but did not abolish tumour-specific antibody production in treated rats. The binding of syngeneic and alloantibodies to surface antigens expressed on hepatoma cells was unaffected by heat treatment. Thus, heat-treated gamma-irradiated hepatoma cells retain a serologically defined tumour-specific antigen but are unable to elicit immunoprotection. By examining the incorporation of radioactive precursors into DNA, RNA and protein in heat-treated cells, it was determined that above 41 degrees C there was a significant decrease in metabolic activity. It is postulated that for the effective induction of transplantation immunity to tumours, tumour-specific antigens should be present on the surface membranes of a metabolically active cell. This hypothesis accounts for the absence or marked reduction of immunoprotection induced by inviable or glutaraldehyde-treated cells, isolated cell membranes and soluble tumour extracts which retain serologically defined tumour-specific antigens.


TNP-470, a synthetic analogue of fumagillin first isolated from Aspergillus fumigatus, is known to be a potent anti-angiogenic compound. The combined effects on tumour growth and tumour angiogenesis of TNP-470 and hyperthermia were investigated. The tumour used was SCCVII carcinoma of the C3H/He mouse. The tumour response was evaluated by the tumour growth (TG) time assay. The TG time is the time required for one-half of the treated tumours to reach three times the initial tumour volume. Significant delay of tumour growth was observed by TNP-470 alone (100 mg kg-1 x 2 or x 4), indicating that TNP-470 alone has antitumour effect in vivo. When TNP-470 (100 mg kg-1 x 2 or x 4) was administered after hyperthermia at 44 degrees C, the TG times of the combined treatment were significantly longer than those of heat alone (44 degrees C) or TNP-470 (100 mg kg-1 x 2 or x 4) alone. However, the TG time of combined treatment with TNP-470 and hyperthermia at 42 degrees C was quite similar to that of TNP-470 alone. This conflicting result on the combined effect of TNP-470 and hyperthermia may be related to the temperature-dependent vascular damage by hyperthermia. Dose-dependent inhibition of angiogenesis by TNP-470 was demonstrated in microangiograms obtained 4 days and 7 days after hyperthermia (44 degrees C for 30 min). It is, thus, suggested that the combined effect of TNP-470 and hyperthermia is attributable to the inhibition of angiogenesis by TNP-470 following heat-induced vascular damage.


The paper considers the stage of spread at the time of operation, the histological type of the tumour, its grade and in particular its mitotic activity, which proved a significant feature


LM985 has been shown previously to hydrolyse to flavone acetic acid (LM975) in mouse plasma and to produce significant anti-tumour effects in transplantable mouse colon tumours (MAC). It has undergone Phase I clinical trials and dose limiting toxicity was acute reversible hypotension. Substantially higher doses of LM975 can be given clinically without dose limiting toxicity. We have investigated the activity of LM975 against a panel of MAC tumours and also the in vitro cytotoxicity of both LM985 and LM975 in two cell lines derived from MAC tumours. LM985 is considerably more cytotoxic than LM975 in vitro but increased length of exposure to LM975 results in improved activity. Single in vivo injection of LM975 showed no activity against the ascitic tumour MAC 15A, moderate activity against the s.c. poorly differentiated tumour MAC 13 and produced a significant growth delay in the well differentiated MAC 26. These latter responses were considerably enhanced by repeated injection 7 days later. Pharmacokinetic studies in mice following i.p. injection of LM985 demonstrated rapid degradation of LM985 to LM975 in the peritoneum. Length of exposure as well as drug concentration appear important factors in determining anti-tumour responses.


Mouse tumour cells were treated with various chemical modifiers. The number of modifying groups per cell was determined with labelled reagents. The effects of the different modifying groups on the immunogenicity of the tumour cells was tested in syngeneic mice for tumour protection using a challenge dose of viable cells at 1000 or 10,000 time LD100. Best protection was obtained after immunization of animals with tumour cells modified with dimethylsulphate or acetic anhydride, or with glutardialdehyde-fixed cells treated with a carbodiimide and methylamine. Up to 40% of the animals remained tumour-free. The other animals exhibited a greatly increased mean survival time. The post-challenge sera showed no detectable amounts of antibodies against the tumour cells.


The MAC 16 is a transplantable murine carcinoma of the colon producing extensive weight loss in tumour-bearing animals. The weight loss is proportional to the size of the tumour and occurs without a reduction in food intake when compared with non tumour-bearing control mice. Weight loss produced by the MAC 16 tumour is accompanied by hypoglycaemia which becomes more extensive as the tumour mass increases. In order to understand the mechanism of the cachexia produced by the MAC 16 tumour the rate of substrate utilization and CO2 formation from both glucose and palmitate has been compared in vitro, with other colon carcinoma cell lines known not to produce cachexia as well as a range of murine and human tumour cell lines. The rate of glucose consumption, lactate production and CO2 formation from both glucose and palmitate is much higher for the MAC 16 than for the other tumour cells. For all cell lines in vitro the consumption of glucose exceeds that of palmitate by a factor of 10(3). Excessive consumption of glucose by the MAC 16 tumour may account for the hypoglycaemic effect on the host. The level of 3 oxo acid CoA transferase, an initiator of ketone body utilization, was found to be much lower in the MAC 16 tumour than non-involved colon. This suggests that the tumour may not be able to metabolize ketone bodies effectively.


Through signals transduced upon integrin ligation by ECM proteins or immunoglobulin superfamily molecules, this family of proteins plays key roles in regulating tumour growth and metastasis as well as tumour angiogenesis. Several integrins play key roles in promoting tumour angiogenesis and tumour metastasis. Antagonists of several integrins ( Antagonists of these integrins suppress cell migration and invasion of primary and transformed cells and also induce apoptosis of primary cells. Integrin antagonists also block tumour angiogenesis and tumour metastasis. Currently, humanised antibody antagonists of integrins α5β1 and αvβ3 as well as peptide inhibitors of integrins αvβ3/αvβ5 are under evaluation as angiogenesis-inhibiting therapeutics in cancer clinical trials.During the last 10 years, novel insights into the mechanisms that regulate cell survival as well as cell migration and invasion have led to the development of novel integrin-based therapeutics for the treatment of cancer. Several integrins play important roles in promoting cell proliferation, migration and survival in vivo controls embryonic development, angiogenesis, tumour metastasis and other physiological processes , others can bind several ligands . Several integrins recognise the tripeptide Arg–Gly–Asp , whereas others recognise alternative short peptide sequences . Inhibitors of integrin function include function-blocking monoclonal antibodies, peptide antagonists and small molecule peptide mimetics matrix  of primary cells such as fibroblasts  also inhibits survival by impairing normal integrin-mediated survival signalling . Additional studies suggest that unligated integrins activate membrane-associated protein kinase A (PKA), which itself can activate caspase 8 in endothelial cells . A cyclic peptide inhibitor of integrin αvβ3/αvβ5, Cilengitide, is in Phase I/II trials for glioblastoma and other cancers (α5β1- and αvβ3-blocking peptides with antitumour angiogenesis and tumour metastasis activities are currently in preclinical development (Several integrin inhibitors are currently under investigation as therapeutics for cancer. Antibody and peptide inhibitors of integrins r cancer , while a cancers . Other pelopment , these iαvβ3, α5β1, αvβ5 and α6β4 show great promise as potential inhibitors of tumour growth and metastasis as well as tumour angiogenesis. Clinical trials are currently underway to evaluate inhibitors of integrin αvβ3, αvβ5 and α5β1 for their usefulness in the treatment of cancer.The studies reviewed here indicate that integrin promote cellular migration and survival in tumour and primary cells. Antagonists of integrins


The effect of the Walker 256 rat carcinoma, growing at a site remote from the liver, on liver glycogen synthesis has been determined. Although the mechanism has not been defined it has been shown that the deposition of liver glycogen in response to a glucose load is inhibited in tumour bearing rats. It has been further demonstrated that, by comparison with the normal liver, the liver depleted of glycogen is more susceptible to the development of metases from circulating cancer cells.


It has been suggested that tumour development and immunocompetence are affected by the menstrual and the oestrous cycle, and sex hormones have been shown to modulate lymphokine production, neuroendocrine activity and immunity. In this study, we assessed natural killer cell activity and host susceptibility to metastasis during the oestrous cycle in the Fischer 344 inbred rat strain. Females were inoculated intravenously with MADB106 tumour cells, a syngeneic mammary adenocarcinoma cell line that metastasises only to the lungs. The susceptibility to metastatic development of this tumour was found to be significantly higher during pro-oestrus and oestrus than during metoestrus and dioestrus. Two days of exposure to oestradiol benzoate caused similar effects in ovariectomised females, and a single administration of progesterone reduced this effect of oestradiol to a statistically non-significant level. The tumour was found to be negative for oestradiol receptors, and its in vitro proliferation rate was not affected by oestradiol or progesterone, suggesting that the effects of sex hormones on the metastatic process are not attributable to a direct effect on tumour cells. Because the metastatic process of MADB106 tumour cells is known, and confirmed here, to be highly controlled by large granular lymphocyte/natural killer (LGL/NK) cell activity, we assessed their role in mediating the effects of the oestrous cycle. The number and activity levels of circulating blood LG/NK cells (NKR-PI+ bright) were studied. Findings indicated oestrous-dependent alterations in the number of LGL/NK cells and suggested a diminished NK activity per LGL/NK cell during pro-oestrus/ oestrus, the same phases that were characterised by higher susceptibility to metastatic development. These findings provide the first empirical evidence for a causal relationship between a short-term exposure to elevated oestradiol/low progesterone levels and decreased resistance to tumour metastasis, and it is hypothesised that an alteration in LGL/NK cell activity underlies these effects. Homologies and relevance to clinical phenomena are discussed.


Norman Rockwell, North America's most beloved and certainly best known illustrator, favored scenes of everyday life and reveled in his ability to tell stories. His Dickensian view of life drove him to paint the world as he would like it to be—no drunken fathers or self-centered mothers, only kindly doctors, duty-bound soldiers, and regular folks at their daily occupations. In his pictures, the sadness was wistful and the problems humorous. Rockwell, who presented himself as an illustrator rather than a fine arts painter, was also an interpreter of the classics and a recorder of history and the contemporary scene, from the Nuclear Test Ban Treaty to the Civil Rights Movement .The realism in Rockwell's illustrations was not photographic. Along with the artful detail, his cast of characters  was loaded with nuances bestowed by the illustrator's genius. The characters sparkled, glowed, and communicated directly with the audience. And the message they sent was exactly the one Rockwell intended the audience to receive. Very much in touch with his surroundings, the artist lived many of the situations that eventually became the subjects of his pictures. He painted what he knew to be the life of a child, a student, a soldier, or a workingman, whether in the Mamaroneck, New York, of his youth, or anywhere else he lived after that. As a result, his characters were universal and accessible to the average viewer.Rockwell's brief experience with the U.S. Postal Service, which may have colored his many depictions of postal workers of all ages, was in the eighth grade. To raise money for art school tuition, he bought the mail route to exclusive Orienta Point from another boy for $25. The wealthy residents of the Point each paid the mail carrier 25 cents to deliver the mail because the regular carrier did not deliver that far from town. Every morning at 5:30, rain or shine, Norman bicycled to the post office, loaded the mail into a leather shoulder bag, and rode 2.5 miles to the end of the Point, delivering the mail to homes on the way.When "Postman Reading Mail" first hit the stands on the cover of the Saturday Evening Post, thousands of letters from postal workers protested the nosy behavior ascribed to one of their own. Rockwell fielded the protests graciously, explaining that the post office was a small operation, in a tiny town, with a few boxes of mail to sort, and the postal clerk had succumbed to boredom and human curiosity: "…if you are interested in the characters that you draw, and understand them and love them, why the person who sees your picture is bound to feel the same way." .If premier illustrator Norman Rockwell were alive today, he would be painting a different mail scene from the one featured on the cover of the Saturday Evening Post in 1922 and now on the cover of Emerging Infectious Diseases. Under the current circumstances of the world, in which the routine and harmless activity of sorting and delivering the mail was deliberately contaminated with a dreaded disease, this fine recorder of history would probably forego the humor in the scene.


A multimeric peptide, Ac-Y16, consisting of 16 YIGSR sequences from laminin was evaluated for its effect on experimental metastasis, angiogenesis and tumour growth of HT1080 human fibrosarcoma cells. Co-injection of 0.5 mg per mouse of Ac-Y16 i.v. with HT 1080 cells inhibited lung colonisation by 100%, whereas 0.5 mg per mouse of monomeric Ac-YIGSR-NH2(AcY1) inhibited by 94%. Ac-Y16 did not show any direct cytotoxicity in tumour cells in vivo. The effect of the peptides on angiogenesis and tumour growth respectively were evaluated by counting areas of neovessels and weighing tumours after the s.c. implantation of HT1080 cells with basement membrane extracts and the peptide into nude mice. Co-injection of 0.5 mg per mouse of AC-Y16 s.c. with HT1080 cells inhibited angiogenesis and tumour growth by 92% (P<0.05) and 76% (P<0.05) respectively, whereas 0.5 mg per mouse of monomeric Ac-YIGSR-NH2(Ac-Y1) inhibited angiogenesis and tumour growth by 40% (P<0.05) and 9% (P>0.05) respectively. It can be inferred from these data that anti-tumour effects of Ac-Y16 are likely to result from anti-angiogenesis. Intraperitoneal administration of Ac-Y16 was also effective in inhibiting angiogenesis, tumour growth and lung colonisation of HT1080 cells. It was concluded that the multimeric YIGSR-containing peptide, Ac-Y16, inhibits angiogenesis, tumour growth and experimental metastasis more than the monomeric form and that it is active when administered i.p., iv. and s.c.


ZD6126 given 2 or 24 h after PTX showed no significant benefit, a result that was attributed to a protective effect of PTX against ZD6126-induced vascular damage and tumour necrosis, a hallmark of VDA activity. Paclitaxel counteracting activity was reduced by distancing drug administrations, and ZD6126 given 72 h after PTX potentiated the VDA's antitumour activity. Schedules with ZD6126 given before PTX improved therapeutic activity, which was paralleled by a VDA-induced increase in cell proliferation in the viable tumour tissue. Paclitaxel given 72 h after ZD6126 yielded the best response (50% tumours regressing). A single treatment with ZD6126 followed by weekly administration of PTX was sufficient to achieve a similar response (57% remissions). These findings show that schedule, sequence and timing are crucial in determining the antitumour efficacy of PTX in combination with ZD6126. Induction of tumour necrosis and increased proliferation in the remaining viable tumour tissue could be exploited as readouts to optimise schedules and maximise therapeutic efficacy.The clinical success of small-molecule vascular disrupting agents (VDAs) depends on their combination with conventional therapies. Scheduling and sequencing remain key issues in the design of VDA–chemotherapy combination treatments. This study examined the antitumour activity of ZD6126, a microtubule destabilising VDA, in combination with paclitaxel (PTX), a microtubule-stabilising cytotoxic drug, and the influence of schedule and sequence on the efficacy of the combination. Nude mice bearing MDA-MB-435 xenografts received weekly cycles of ZD6126 (200 mg kg The tumour vasculature is an established target for the therapy of solid tumours. Agents designed to prevent the formation of new tumour vessels (antiangiogenic therapy) or to damage already formed vessels (vascular targeting/disrupting therapy) have been developed and have shown efficacy in preclinical models, and recently, in clinical studies  , antagonists of junctional proteins, and compounds that target proteins expressed exclusively on the tumour vasculature and are usually used to deliver bioactive molecules  and international laws and policies  Mice were used when 8–10 weeks of age (mean body weight=23±2 g). Nude mice were housed in filtered-air laminar flow cabinets and manipulated following aseptic procedures.Female NCr-nu/nu mice were obtained from the animal production colony of the National Cancer Institute, Frederick Cancer Research and Development Center .  was dissolved in 50% Cremophor EL  and 50% ethanol and further diluted with saline immediately before i.v. administration.ZD6126 (MW 437 Da), provided by AstraZeneca, Alderley Park, Macclesfiled, UK, was dissolved in PBS with 0.5% Na6) were implanted s.c. in the flanks of nude mice . Tumour growth was monitored two times a week by measuring tumour size with calipers, and estimating tumour weight, in grams, calculated as ((length × width2)/2). Treatment started when tumours reached the size of approximately 450 mg, since preliminary results indicated that the activity of VDA is optimal on established tumours (data not shown). Animals were randomised on the basis of tumour weight and subjected to treatment (each group consisted of 6–10 mice). Mice received ZD6126 (i.p.) and PTX (i.v.) at the doses, schedules, and sequence detailed in the Results section. Control mice received the corresponding vehicle. Experiments were concluded when tumours reached a median weight of 2±0.5 g or 4–5 weeks after the last treatment.MDA-MB-435 cells  × 100). Treatment was considered active when optimal %T/C  was lower than 40%. Specific growth delay (SGD) was calculated as ((DT treated – DT control)/DT control), where DT (doubling time) is the time (in days) necessary to double the initial tumour weight (namely to reach a RTW=2). Specific growth delay was considered active when >1. Complete regression was defined as fully regressed tumours, yet undetectable by palpation at the end of the experiment. Statistical differences among groups were evaluated by ANOVA followed by Bonferroni/Dunn post hoc test.Tumour growth was expressed as relative tumour weight RTW=−1, i.v.), followed 2, 24, 72 h, and 1 week later by ZD6126  (n=5 mice per group). Tumours were then removed 24 h after treatment with ZD6126, cut sagittally along the midline, fixed in formalin and processed for histological analysis. Five-micrometer sections were cut and stained with haematoxylin and eosin (H&E) following standard procedures. Images of the whole sagittal H&E-stained tumour sections were captured using a Nikon Super Coolscan 4000 ED film scanner . Necrosis was analysed by computerised image analysis , exploiting the difference in staining intensity between vital and necrotic tissue  or vehicle (n=5 mice per group), fixed and stained with H&E as above or immunostained for Ki-67. Antibodies against Ki-67  were used according to the manufacturers' specifications. Negative control included omission of the primary antibody and incubation with non-immune serum. Background immunostaining was found to be negligible when non-immune serum was substituted for specific antiserum. Immunostaining was visualised by the avidin–biotin–peroxidase complex method using the Vectastain Elite ABC kit  with 2,2′-diaminobenzidine as a substrate. Haematoxylin was used to counterstain the nuclei of the specimens after immunostaining. Positively stained cells were identified by the presence of a brown nuclear precipitate. Negative cells were identified by the absence of precipitate and only blue counterstain.For the evaluation of ZD6126-induced cell proliferation, MDA-MB-435 tumours were collected 24 h after administration of ZD6126 (200 mg kgpost hoc test (comparison between more than two groups) or Mann–Whitney U-test (comparison between two groups).For each H&E- and Ki-67-stained section, five random non-overlapping fields at a magnification of × 400 (high-power field (HPF)) in the viable areas of tumours were captured with an Olympus Camedia C-3030 Zoom digital camera . Images were analysed with Image Pro-Plus. Mitotic figures and total cell number were counted in five HPFs/tumours, and the mitotic index was expressed as the number of mitoses per 500 cells. The proliferation index was expressed as the percentage of Ki-67-positive nuclei per total number of cells. Statistical differences among groups were evaluated by ANOVA followed by Bonferroni/Dunn We first evaluated the effect of combination regimens with ZD6126 given after PTX. The rationale for this schedule was that the vascular effect of the VDA would cause ‘trapping’ of the already present cytotoxic drug within the tumour, and, at the same time, that the possible VDA-induced impairment of PTX distribution in the tumour would be avoided  modestly affected the tumour growth with a %T/C ranging from 68.7 to 76.7. The administration of ZD6126 2 h after PTX treatment showed no significant additional benefit compared with PTX alone. This was evident at all doses of PTX .ectively . ZD6126 The lack of potentiating effect of this combination schedule is in agreement with the reported interference between the two agents, which have an opposite effect on the same target, that is, microtubules (−1) 2 or 24 h before the VDA  and efficacy . Specific growth delay was 2.1 and 3.0 for PTX and the combination, respectively , tumours in all mice treated with PTX alone eventually regrew, and only in the group receiving the ZD6126/PTX combination were complete remissions observed (three out of six).The potential drawback of this schedule is that pretreatment with the VDA impairs the functional properties of the tumour vasculature, hence possibly affecting the distribution of PTX within the tumour. Since the activity of this class of molecules, including ZD6126, is rapid and reversible  24 h after its administration. Accordingly, the administration of PTX 24 h after ZD6126 was associated with an increased antineoplastic activity of the combination, suggesting that the mechanism of the favourable effect of pretreatment with ZD6126 might be the increment in the number of proliferating, PTX-responsive cells in the tumour periphery. Moreover, a preliminary pharmacokinetic analysis showed that tumours pretreated with ZD6126 presented an increased ratio between PTX at the tumour periphery and in the inner part of the tumour, suggesting that in VDA-treated tumours the chemotherapeutics concentrate where the tumour is more viable.The actual target cell in the tumour periphery is still unidentified. Besides tumour cells, endothelial cells might also be targeted by the combination, as shown by the increase in endothelial cell apoptosis induced by the combination of ZD6126 with cisplatin . HoweverOur observation that an enhanced combination schedule was obtained by increasing the interval between administration of ZD6126 and of the chemotherapeutic  might indicate that at these conditions a trade-off is achieved between the ‘positive’ effects of VDA on cell proliferation and/or CEP recruitment at the tumour periphery and the ‘negative’ effects of ZD6126 on the distribution of PTX within the tumour. The final outcome of combinations of cytotoxic agents with VDA is therefore the result of each drug's effect on tumour cells and on host tissue compartments (such as tumour blood vessels), and depends on their pharmacological interactions. All these factors account for and should be considered when attempting to optimise schedules and sequence.Clinical and preclinical studies with ZD6126, as well as with other VDAs, indicate that these compounds are active at well-tolerated doses, well below the MTD. The toxicity profile differs from that observed with cytotoxic chemotherapeutics, indicating that additive toxicity should not be an issue of concern in combination regimens (In conclusion, our study indicates that sequencing is crucial in determining the antitumour efficacy of chemotherapy with VDA, and that scheduling of the interval between treatments might be relevant in determining the magnitude of the response. In the case of combinations of tubulin-binding compounds, such as ZD6126 and PTX, VDA-induced necrosis and proliferation index can been used as biological readouts to optimise scheduling of treatments. The best response was obtained by giving the VDA before the tubulin-binding cytotoxic agent and by allowing a significant time interval between the two agents.


The subcutaneous growth of 2 antigenically distinct syngeneic methylcholanthrene-induced murine fibrosarcomas, designated H1 and H7, were significantly augmented by the concomitant administration of E. coli endotoxin (LPS). Amounts as little as 0.2 micrograms i.p. potentiated tumour growth. The weakly antigenic tumour, H1, was more susceptible to provocation by LPS than the more strongly antigenic H7. Maximum provocation of H1 tumour growth occurred when LPS was injected 1 day before the administration of 5000 tumour cells. In contrast, significant anti-tumour resistance resulted if LPS was administered 6 days before the inoculation of tumour cells. Preliminary evidence indicates that low doses of LPS can facilitate the "sneaking through" phenomenon. Enhancement of tumour growth could not be demonstrated with sera or plasma from tumour-bearing mice, unless the samples were contaminated with endotoxin. The results illustrate the importance of excluding endotoxin from solutions used in studies of experimental tumours.


The strategy of combining carbogen breathing and nicotinamide to overcome chronic and acute hypoxia respectively is being evaluated clinically. The effects of both agents individually and in combination on relative perfusion of 400-700 mm3 RIF-1 tumours and normal tissues were measured by 86Rb extraction. Carbogen breathing alone for 6 min increased relative tumour perfusion by 50-70% compared with control at flow rates of 50 to 200 ml min-1, but the effect was lost at 300 ml min-1. All flow rates also produced similar increases in relative perfusion of lung, of between 36% and 58%, and smaller increases in skin, of between 20% and 34%. The minimum breathing time at 150 ml min-1 to produce a significant increase in relative tumour perfusion was 4.5 min, and the effect was maintained up to 9 min. Nicotinamide alone at 1000 mg kg-1 60 min before assay did not alter relative tumour perfusion. Comparing the combination of nicotinamide with 6 min carbogen breathing at 150 ml min-1 with carbogen breathing alone showed no difference in relative tumour perfusion; increases were of 36% and 42% respectively. Nicotinamide-induced alterations in microcirculation associated with reduction of acute hypoxia have therefore not been detected by 86Rb extraction. The perfusion-enhancing effect of carbogen in this tumour is probably an important component of its radiosensitising ability, in addition to its known ability to increase the oxygen-carrying capacity of the blood, and should be taken into consideration in clinical studies.


Metastatic spread of some solid tumours is thought to depend upon the adhesion of tumour cells to the vascular endothelium followed by extravasation into surrounding tissues. We investigated the role of beta 1 integrins in the adhesion of the breast adenocarcinoma cell line MDA-MB-231 and the melanoma cell line RPMI-7951 to quiescent human umbilical vein endothelial cells (HUVEC) in vitro. In the course of adhesion assays, tumour cells were observed to adhere to quiescent HUVEC monolayers, particularly at endothelial cell-cell junctions. Immunohistochemistry revealed concentration of beta 1 integrin expression at these sites. Adhesion was reduced by pretreatment of either tumour cells or HUVEC with antibodies against beta 1 integrins. Simultaneous treatment of HUVECs and tumour cells with these antibodies produced an additive blocking effect, consistent with a heterotypic adhesion mechanism. Our data suggest that tumour cell and endothelial beta 1 integrins may play a crucial role in the arrest and migration of tumour cells through the vascular endothelium in the absence of endothelial 'activation'.


Nerve growth factor (NGF) administration to athymic mice with transplanted human bromocriptine-resistant prolactinoma, results in the expression of dopamine D-2 receptors in the tumour and restores sensitivity to subsequent treatment with bromocriptine, which then produces normalisation of plasma prolactin and tumour regression. Sequential administration of NGF and bromocriptine thus may be a promising therapy for patients refractory to bromocriptine.


Groups of hairless mice were treated with 4 skin applications of 470 nmol 3-methylcholanthrene (MCA) in benzene and 4 of 20 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) in various sequences, twice a week, together and separately. Three days after the last application, cell kinetic investigations were made comprising the counting of basal and suprabasal cells, the assessment of hyperplasia, the mitotic rate by the stathmokinetic method, the labelling index and the specific activity of DNA after injection of a dose of [3H]dT, and the determination of percentage of cells in each cell-cycle phase by flow cytometry. These studies showed that various treatment schedules with 4 applications stimulated proliferation and caused epidermal hyperplasia, but there was no significant difference between the groups in degree of growth stimulation. There was a significantly higher tumour production by all the combinations than by MCA alone. It was of no significant importance for the tumour production whether the 4 applications of MCA came before or after the 4 of TPA. Alternating treatment  seemed to give a higher tumour risk than the other treatment sequences. The consequences of these results for the two-stage theory of carcinogenesis (stating that initiation must come first) are discussed, and it is concluded that  initiation does not need to come first for a good tumour yield.


Here we report the case of a 54-year old, immunocompetent German patient with primary varicella whose Varicella-Zoster Virus (VZV)-specific T-cell responses could be detected early in infection and before the onset of seroconversion. This case demonstrates that the detection of VZV-specific T-cells may under certain circumstances support the diagnosis of a primary varicella infection, as for example in cases of atypical or subclinical varicella or in the absence of detectable VZV DNA in plasma. Varicella-Zoster virus (VZV) causes varicella during primary infection and may cause herpes zoster after reactivation from latency. Varicella is typically diagnosed by characteristic clinical signs and usually does not require laboratory testing. Due to the introduction of mass vaccination programmes, however, the incidence of typical varicella has declined. This decline has led to a reduced experience of physicians in diagnosing varicella. Furthermore, an increased incidence of atypical and vaccination breakthrough varicella infections has been described -4. For tin house flow cytometry assay after intracellular interferon γ staining of isolated and ex vivo stimulated peripheral blood mononuclear cells (PBMCs). For a detailed protocol see additional file We report the case of a 54-year-old, immunocompetent German (Caucasian) man who presented with skin rash and fever (day 0). Due to the clinical appearance of a typical "varicella-like" rash phenotype, antiviral therapy with Brivudine was initiated at the day of the patient's presentation and rash onset. Serological analysis of blood taken at day 0 exhibited no VZV-specific antibodies indicative for a past (VZV-IgG) or primary (VZV-IgM) VZV infection. In order to confirm the suspicion of primary varicella, a second blood sample was taken at day 2 post rash onset (p.r.o.). Again, subsequent serological testing for VZV-specific IgG and IgM antibodies exhibited negative results. However, abundant numbers of VZV-specific CD4+ T-cells could be detected within this second sample. Analysis of VZV-specific T-cells was performed by a novel The development of novel assays for the detection of virus-specific T-cells will contribute to answer a variety of medically relevant questions that could so far not be addressed sufficiently. Among them (a) the diagnosis of a pathogen involvement if serological parameters are impaired, (b) the differentiation between different stages of viral infections, (c) the monitoring of the immune status of immunosuppressed patients after solid organ or bone marrow transplantation, (d) the examination of vaccine efficiency with respect to cellular immunity, or (e) the determination of immune correlates of protection towards a herpes zoster reactivation as indication for zoster vaccination. Here we report that the detection of VZV-specific T-cells may (f) additionally support the rapid diagnosis of an early varicella infection before seroconversion.As to our knowledge, no systematic comparison between the onset kinetics of seroconversion and occurrence of VZV-specific T-cells within patients at early stages of varicella infection has been performed until today. Seroconversion has been described to appear one to seven days p.r.o. . In contWithin our patient, detectable VZV-specific CD4+/IFNγ+ T-cell titers were highest at day two p.r.o. and before the occurrence of seroconversion. At this time, titers of up to 1% of total CD4+ T-cells were detected. Such elevated CD4+ T-cell titers have been described during acute varicella infections . The perWe conclude that the analysis of VZV-specific CD4+ T-cells might support the rapid diagnosis of primary varicella under certain circumstances, as for example in cases of atypical or subclinical varicella or in the absence of detectable VZV DNA in plasma.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.ex vivo T cell stimulation. LD provided protocol and reagents for the urea treatment of VZV lysates for the ex vivo T-cell stimulation. HC provided the basic protocols for the analysis of virus-specific T-cells and helped interpreting the FACS data. HN performed the real time PCR analysis. GJ provided overall supervision of the project and helped with interpretation of clinical data. All authors read and approved the final manuscript.AB planned the project and wrote the paper. RH performed all FACS analyses for the detection of VZV-specific T cells. JE collected the patient samples. MGVP, K-IP and AP participated in the production of recombinant glycoprotein E for the Protocol for the detection of VZV-specific T-cells: This additional file provides a detailed protocol for the detection of VZV-specific T-cells from heparinized blood.Click here for file