Renal cell carcinoma (RCC) represents one of the most immunoresponsive cancers. Antigen-specific vaccination with dendritic cells (DCs) in patients with metastatic RCC has been shown to induce cytotoxic T-cell responses associated with objective clinical responses. Thus, clinical trials utilizing DCs for immunotherapy of advanced RCCs appear to be promising; however, detailed analyses concerning the distribution and function of DC subsets in RCCs are lacking.We characterized the distribution of the different immature and mature myeloid DC subsets in RCC tumour tissue and the corresponding normal kidney tissues. In further analyses, the expression of various chemokines and chemokine receptors controlling the migration of DC subsets was investigated.The highest numbers of immature CD1a+ DCs were found within RCC tumour tissue. In contrast, the accumulation of mature CD83+/DC-LAMP+ DCs were restricted to the invasive margin of the RCCs. The mature DCs formed clusters with proliferating T-cells. Furthermore, a close association was observed between MIP-3α-producing tumour cells and immature CCR6+ DC recruitment to the tumour bed. Conversely, MIP-3β and SLC expression was only detected at the tumour border, where CCR7-expressing T-cells and mature DCs formed clusters.de novo local expression of SLC and MIP-3β induces accumulation of mature DCs at the tumour margin forming clusters with proliferating T-cells reflecting a local anti-tumour immune response.Increased numbers of immature DCs were observed within the tumour tissue of RCCs, whereas mature DCs were found in increased numbers at the tumour margin. Our results strongly implicate that the distribution of DC subsets is controlled by local lymphoid chemokine expression. Thus, increased expression of MIP-3α favours recruitment of immature DCs to the tumour bed, whereas  DC prog of CCR7 ,8,9. A c of CCR7 , as obse of CCR7 ,6.The involvement of DCs in tumour immunity has clinical importance. The infiltration of DCs into some primary tumour types has been found to be associated with significantly improved patient survival and a reduced incidence of recurrent disease ,12. It iPromising results have been previously reported using DC-based vaccination against immunogenic tumours, such as melanoma or renal cell carcinoma (RCC) ,18. A suMore recently, novel markers have emerged allowing the identification of a broader spectrum of DC subpopulations with respect to their function on formalin-fixed and paraffin-embedded tissue. Thus, we characterized the phenotype, distribution, and maturation of the different DC subsets in RCCs. In addition, we analyzed the local expression of chemokines that are known to play a key role for the recruitment of DC subsets.Tumours and corresponding tumour-free tissue of nephrectomy specimens from 24 patients with RCCs were included in this study. All tumours were freshly obtained from the Urologic Department's operating room. The patient ages ranged from 36-75 years . None of the patients investigated in this study were treated before surgery. The formalin-fixed and paraffin-embedded specimens were cut into 5-μm-thick sections and placed on poly-L-lysine-treated glass slides. Representative sections were stained with routine hematoxylin and eosin (H&E) and evaluated. The histologic types of cancer were as follows: clear cell (n = 17); papillary (n = 4); chromophobe (n = 1); and sarcomatoid renal cell carcinoma (n = 2). This study was approved by the Ethics Committee of Georg-August-University Göttingen, and the Helsinki Declaration regarding the use of human tissue was followed. Informed consent was obtained from the patients for the use of their tissue samples.The antibodies used in the study and the optimal working dilutions are listed in Table For double immunofluorescence staining, the slides were stained for 1 hour with unconjugated primary antibody, followed by incubation with indocarbocyanine 2 (Cy2)-conjugated goat anti-mouse or goat anti-rabbit F(ab)-fragments  at a saturating concentration for 60 minutes. For the first step in immunofluorescence staining for MIP-3α, MIP-3β, and SLC, we adopted the TSA-Kit (NEN) using FITC-conjugated tyramide for fluorescence amplification. Sections were washed and incubated with the second antibody for 60 minutes at room temperature, followed by incubation with indocarbocyanine 3 (Cy3)-conjugated goat anti-mouse or goat anti-rabbit F(ab)-fragments (both from Dianova) for 60 min at room temperature.Confocal fluorescence images were obtained on a Leica TCS  confocal system mounted on an Olympus BX50 WI microscope . Possible cross-talk between FITC or Cy2 and Cy3, which could give rise to false-positive co-localization of different signals, was avoided by careful selection of the imaging conditions.http://www-genome.wi.mit.edu. Optimal conditions for all primers were established by amplifying cDNA samples from human tonsil or lymph node.Sequences of primers used in the study are listed in Table TaqDNA polymerase kit (Qiagen). Expression of MIP-3α, MIP-3β, and SLC was normalized to ß-actin expression to compensate for different sample capacities. Results derived from the PCR standard curve are given in attomoles per μg of total cellular RNA. cDNA from a human lymph node was used as a positive control template for each primer pair. Negative controls with water instead of cDNA were always included.Total cellular mRNA was extracted with the RNeasy Mini Kit (Operon-Qiagen). RNA integrity and quantity was assessed using the Agilent Bio-analyzer 2100 . Reverse transcription with random hexamer primers was performed with the Omniscript RT Kit (Qiagen). Quantification of MIP-3α, MIP-3β, SLC, and ß-actin mRNA expression was performed on an iCycler iQ real-time detection system  using the HotStar 2 in RPMI-1640 containing 10% fetal calf serum, 10 mm l-glutamine, 1% penicillin/streptomycin, 2.5% HEPES buffer, and 1% amino acid solution.The RCC cell lines  were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen . All 3 cell lines were cultivated at 37°C in 5% COThe evaluation of immunoreactivity was performed on sections stained for CD1a and CD83. Because of the heterogeneous distribution of DC, the quantification was performed by the hot-spot method. This method allows quantification of cells in hot spots defined in this study as areas containing the highest density of positive cells. Accordingly, five hot spots per section were counted with an eyepiece graticule at 400x magnification. Statistical analyses were applied using GraphPad Prism . Statistical comparisons were performed applying the unpaired t-test or the Mann-Whitney test; p values < 0.05 were considered as statistically significant.To evaluate the total number of DCs in tumour tissues of RCCs and the corresponding normal kidney tissues, immunostaining for CD11c and Fascin was performed. These markers have been shown to be widely expressed by subsets of immature and mature myeloid DCs . Unfortunately, both markers proved not to be specific for DCs in renal tissues. For instance, immunostaining for CD11c Figure  revealedImmature DCs were characterized by CD1a expression Figure , and theWe next sought to determine if mature DCs were also infiltrating RCC tumour tissues. Mature DCs were characterized by expression of CD83 Figure . CD83+ mBecause mature DCs can normally be found only in secondary lymphoid organs where they closely interact with Ag-specific T cells, we next analyzed the T cell distribution and activation pattern in RCC samples. In 22 of the 24 evaluated samples, CD3+ T cells infiltrated peri-tumoural areas, where they could be seen either scattered throughout the area in close proximity to tumour cells or clustered. Double-staining for CD3 and CD83 demonstrated T cell clusters around mature DCs in the peri-tumoural areas Figure . TypicalTo explore the differential expression of the lymphoid chemokines MIP-3α, MIP3-β, and SLC in RCC cell lines and RCC tissue RT-PCR analyses were performed. RT-PCR on total mRNA isolated from three established RCC lines  showed abundant PCR products for MIP-3α by all three cell lines investigated . For carcinoma-derived mRNA, a statistically significant increased expression of MIP-3α (p < 0.05) mRNA was observed in comparison to mRNA of corresponding normal kidney tissue Figure . In contThe expression of MIP-3α, MIP3-β, and SLC was then evaluated by immunohistochemistry and double-immunofluorescence on paraffin sections of the cases analyzed by RT-PCR. Immunostaining for MIP-3α was observed within the cytoplasm of tumour cells in all cases investigated Figure . FurtherImmunohistochemistry for MIP-3β and SLC demonstrated no staining of epithelial cells, including tumour cells and normal kidney tubules. In addition, no staining was observed by tumour-associated macrophages or other inflammatory cells, such as CD3+ T-cells or CD79a+ B-lymphocytes. In contrast, an increased number of MIP-3β-expressing cells were observed within the T-cell clusters Figure . The celCCR6-positive cells showing dendritic morphology were randomly distributed throughout the tumour bed Figure  in all cIn contrast, lymphoid aggregates of the tumour border demonstrated a strong expression for CCR7 Figure . Within in vitro-generated DCs, which have been fused with tumour cells or loaded with tumour antigens [A RCC is considered to be one of the most immunoresponsive cancers in humans. During the last few decades, promising new immunologic-based treatment strategies for this tumour entity have been developed . Howeverantigens -21.in vivo is required for development of an optimal vaccination strategy. One very important issue in this context represents a detailed knowledge concerning the type and distribution of DC subsets, as well as the mechanisms underlying their migration, maturation, and function in RCCs. Recent data point to a significant role of DC subsets in Th1/Th2 polarization and the induction of the tumour immune response [Different approaches of RCC vaccines have been explored in the metastatic and adjuvant setting in several studies ,23. Reviresponse ,43. Thusresponse . Thereforesponse ,15,45,46in vitro and in vivo to express CD1a, but not Langerin, we currently conclude that CD1a+/Langerin- tumour-infiltrating DCs represent immature interstitial DCs. The significance of this heterogeneity remains to be established. Earlier studies revealed that interstitial DCs generated in vitro from CD34+ precursors display more potent phagocytic activity than LCs [All tumour samples displayed variable immature DC infiltration. Tumour-infiltrating immature DCs were heterogeneous, and two populations were identified. One population represented the CD1a+/Langerin+ Langerhans-cell type and the other type was CD1a+/Langerin- non-Langerhans cell. Since dermal  DCs were found The observed numbers of immature CD1a+ DCs in the tumour environment was much higher than in normal kidney tissues, suggesting increased homing and infiltration of immature DCs by the tumour. This finding was also been described by Troy et al. ,48 nearlHowever, in contrast to the observed immaturity of intratumoural DCs, the more important finding might be the presence of numerous mature DCs accumulating specifically within the peri-tumoural areas. This finding suggests that additional factors might be involved in mature DC distribution. Because mature DCs are typically observed in lymphoid organs, where they closely interact with T cells, it is tempting to consider that their presence within the tumour tissue might reflect an ongoing tumour-specific immune response. Thus, the mature DCs could derive from any of the immature DC subset discussed above within the tumour tissues. The peri-tumoural localization of mature DCs observed in our study corresponds to observations in other tumour types, and RCC as well ,47. The de novo development of a tertiary lymphoid tissue at the invasive margin of the tumours. Our results show a significant increased expression of lymphoid chemokines (SLC and MIP-3β) at the tumour border in contrast to tumour and normal kidney tissues. These results imply that the increased expression of SLC and MIP-3β may lead to a chemokine microenvironment normally observed in secondary lymphatic tissues. Expression of these chemokines favours homing and interaction of CCR7-expressing cells, such as mature myeloid DCs and naïve or memory T cells, facilitating their interaction for an optimal anti-tumour immune response. Thus, in analogy to secondary lymphoid organs, such as lymph nodes, chemokine-dependant co-localization of T-cells and antigen-presentation occurs at the tumour border, leading to the local generation of anti-tumour-specific T cells.What instigates the mechanisms underlying the accumulation of mature DCs with a prominent T cell response in RCCs. Our results support the concept that specific homing of CCR7+ leucocytes to the tumour border may contribute to the anti-tumour response in RCCs, which at least partly resembles Interestingly, various studies have shown that the presence of a dense DC infiltration has been associated with prolonged survival and reduced incidence of metastases in patients with various human cancers, such as colorectal, gastric, esophageal, oral, and lung carcinoma -59. In bThese and our observations suggest that the presence of an "immunologic functional unit" in RCCs consisting of antigen-presenting DCs and activated T cells at the tumour border might represent prognostic significance. In this scenario the combination of a low number or absence of DCs with a low density of activated T cells would predict a worse outcome, while a high density of mature DCs and activated T cells predicts a favourable disease outcome. This would also suggest that the presence of mature antigen-presenting DCs at the primary site together with that of activated T cells, represents a functional immune response against RCC progression. Thus, our observations open new insight regarding DC involvement and function concerning the anti-tumour response in RCCs. However, the limited number of RCC tumour samples analyzed in this study and the short follow-up times does not allow us to establish a prognostic significance of the infiltration of tumour by immature or mature DCs. Such determination will require the analysis of a large number of samples in prospective or retrospective studies. Therefore, further analysis of the APC system will provide clues to the understanding of tumour immunity as well as improvement of the efficiency of vaccine strategies in RCCs and other immunogenic tumour entities.RCC: renal cell carcinoma; DC: dendritic cell; APC: antigen-presenting cellThe authors declare that they have no competing interests.PM performed the design of the study and the statistical analysis. PM and SB performed the experimental investigations. WM participated in the writing of the manuscript. HJR participated in conception of the idea and writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/578/prepub


They cause complete regressions of some established subcutaneous (s.c.) or intradermal (i.d.) tumours but not of intraperitoneal (i.p.) tumours when administered either systemically or directly into the tumour. To achieve this effect the tumours must be fully established and the best results were obtained when treatment was started 7 days after transplant. If treatment is started within the first 3 days following the transplantation of the tumour then only a slight inhibition of growth rate was observed. These agents can also act prophylactically and protect mice against a subsequent challenge but only if this is given i.p. and not if given s.c. or i.d. The prophylactic action is explained by the action of dsRNA and endotoxin on peritoneal macrophages which cause them to become cytotoxic to tumour cells .Double stranded RNA (dsRNA) whether isolated from a fungal virus or prepared synthetically  but is not itself sufficient. Immunosuppression by whole body irradiation or by antilymphocyte serum also interferes with the antitumour action of dsRNA and endotoxin in spite of the fact that haemorrhagic necrosis still occurs. Also, the magnitude of the antitumour action correlated in a series of different tumours with their antigenicity. The observed tumour regressions are probably brought about by (1) vascular damage in the tumour which permits immune defence mechanisms of the host to gain access to the tumour and (2) activation of macrophages present within the tumour. The relative contribution of these two mechanisms may depend on the nature of the tumour and the route of administration of the active agents.The therapeutic effect of systemically administered endotoxin and dsRNA on established tumours is not the result of a direct action on the tumour cells themselves but is a complex process requiring the co-operation of several host factors. Haemorrhagic necrosis involving coagulation is essential (Dibenyline, which protects against the lethal action of endotoxin by preventing the action of the catecholamines on the α-adrenergic receptors, makes it possible to increase the effectiveness of endotoxin in tumours by allowing a large dose to be given. Lipid A, a derivative of endotoxin which does not contain polysaccharide, has similar antitumour action to dsRNA and endotoxin. Some common features of the chemical structure of lipid A and dsRNA are discussed.


Myolipoma is a rare, benign, lipomatous tumour which most commonly occurs in the retroperitoneum, pelvis, and abdomen. A 4-year-old boy presented with a painless enlarging mass in the left paraspinal region. Magnetic Resonance Imaging (MRI) revealed a soft tissue lesion with high fat content and areas of calcification. Excision and histopathological analysis revealed a tumour composed of lobules of mature adipose tissue and broad septa of well-differentiated smooth muscle tissue. The smooth muscle nature of the nonfatty component was demonstrated by a diffuse and strong immunoreactivity for smooth muscle actin and desmin. The mass was reported as a myolipoma. The patient made an unremarkable recovery from surgery and remains healthy with no signs of recurrence at seven years. This paper represents the youngest patient diagnosed with this rare soft tissue tumour which is normally confined to the adult population. A newly reported site of the tumour is also highlighted. Myolipoma is an extremely rare benign tumour related to lipoma. It is composed of a mixed proliferation of adipose and smooth muscle tissues presenting, in most patients, as a large painless mass. The tumour is benign with no reported cases of recurrence or metastasis, suggesting that complete surgical excision is curative. Most cases of this tumour have been reported in the retroperitoneum, pelvis, and abdomen. There are fewer reports of the tumour affecting other locations. We present a case of myolipoma affecting the erector spinae muscle group. As far as we are aware, this is the first reported case of the tumour in this location. Furthermore, this case represents the youngest patient diagnosed with a myolipoma.A four-year-old male was referred to our institution with a two-year history of a painless swelling on the left side of the back. The mass had been slowly increasing in size with the child's growth. The patient was otherwise healthy. Examination revealed a firm nonfluctuant, nonpulsatile mass in the left paraspinal region. No tethering or overlying skin changes were noted. Further examination was unremarkable. Investigations included ultrasound, computed tomography, and MRI. The ultrasound identified a tense cystic paraspinal mass. Magnetic resonance imaging (MRI) was performed and demonstrated a well-defined intramuscular mass overlying the lower thoracic spine. The mass was of fat signal intensity with multiple large areas of low signal intensity in keeping with calcification. No involvement of the posterior spinal elements or intraspinal extension was noted Figures . A CT scThe patient attended for excision biopsy and an ovoid tumour (6.3 × 3.9 × 2.6 cm) was resected. Macroscopically, the mass appeared covered by a thin layer of skeletal muscle. Histologically, the tumour was composed of lobules of adult-type mature adipose tissue separated by broad septa of well-differentiated smooth muscle tissue. This was characterised by elongated spindle cells with cigar-shaped nuclei and eosinophilic cytoplasms, arranged with a fascicular growth pattern . Both coThe patient made an unremarkable recovery from surgery and remains healthy with no signs of recurrence.Myolipoma was first described by Meis and Enzinger in 1991 , orbit , pericarPresentation tends to be with an enlarging painless mass. Most reported cases describe large tumours averaging 16 cm in the greatest dimension . It is mThere is paucity of information regarding the imaging of the lesion. Ultrasound may demonstrate a heterogeneous mass . In uterMacroscopically, the tumour is either partially or completely encapsulated and upon sectioning demonstrates a yellow/white cut surface . HistoloThe differential diagnosis is important due to the number of lipomatous tumour variants . DediffeSurgical excision is the treatment of choice for these lesions. To date, there are no reports of recurrence and the complication rate is minimal.This case represents an exceedingly rare and interesting benign tumour. Surgeons and histopathologists should be aware of the lesion in order to correctly diagnose and treat it. We report a new location for the tumour occurring in the youngest patient diagnosed so far. Despite the benign nature of the tumour, radiological investigations may indicate a more sinister diagnosis. Therefore, excisional biopsy in a specialized centre, where the necessary expertise is available, is recommended.


We have used the nitrosomethylurea-induced rat mammary tumour model to study the effects of parenteral administration of human recombinant tumour necrosis factor (rHu-TNF) and rat gamma interferon (IFN-gamma). An inbred strain of tumour bearing female Ludwig/Wistar/Olac rats were randomised to either treatment or control groups. Two independent studies showed that combined treatment with rHu-TNF and rat IFN-gamma induced significant tumour regression over 4 weeks . Treatment with either rHu-TNF or rat IFN-gamma given individually did not affect the overall rate of tumour growth (P = 0.157 and 0.40 respectively) although an initial reduction in tumour size was observed during the first few days after injection. Measurement of circulating oestradiol levels in groups in which maximum tumour regression was observed showed no statistically significant difference when compared to the control group. Similarly, measurement of oestrogen receptor content showed no statistically significant difference between rHu-TNF-gamma or rat-IFN-gamma treatment or combined treatment of rHu-TNF and IFN-gamma with the control group. We conclude from these observations that combined therapy with rHu-TNF and rat IFN-gamma may prove to be useful new forms of treatment for human breast cancer.


Pancreatic cancer is difficult to treat, even for tumours localized to the pancreas. Photodynamic therapy (PDT) is a non-thermal technique for producing localized tissue necrosis with light after prior administration of a photosensitizing drug and it could have a role in the local treatment of these cancers. We studied PDT in a transplanted cancer in the hamster pancreas using the photosensitizer mTHPC (meta-tetrahydroxyphenylchlorin). Fluorescence microscopy showed maximum levels of mTHPC in normal pancreas 2-4 days after sensitization and in tumour at 4-5 days. For PDT, animals were given 0.1 or 0.3 mg kg(-1) mTHPC and the tumour was treated at laparotomy 2 or 4 days later with red light  delivered via a single fibre touching the tumour surface. The maximum zone of tumour necrosis (seen 3 days after PDT) was 8.7 mm in diameter with continuous irradiation, increasing to 12.4 mm with light fractionation . The main complication was sealed duodenal perforation, seen in 3 of 16 animals, probably due to inadequate shielding of the duodenum from the light. The duodenal problems seen in hamsters are unlikely to cause trouble in the much thicker human duodenum. PDT tumour necrosis in this animal model has now been shown with a range of photosensitizers, but mTHPC is attractive as it is likely to produce the largest volumes of necrosis around each treatment point with short light exposure times. This technique could have a role in the treatment of localized cancers of the pancreas in patients unsuitable for surgery and can now be considered for preliminary clinical trials.


Reticuloendothelial (RE) phagocytic and circulating plasma opsonic activity was evaluated in rats transplanted with the Walker 256 carcinoma tumour in an attempt to evaluate the role of opsonic protein in governing the functional state of the macrophage system. Animals transplanted intramuscularly with 2 X 10(4) viable tumour cells manifested 2 peaks of RE stimulation at 6 and 14 days post-transplantation with a subsequent decline in the phagocytic activity over the 14-30 day period. Increased phagocytic activity as determined by colloid clearance was primarily a reflection of hepatic Küpffer cell hyperphagocytosis while the decline in phagocytic activity was related to a decrease in Küpffer cell function. The initial peak of RE stimulation was associated with an elevation in the blood opsonin level and no significant enlargement of the liver and spleen. In contrast, the second peak of RE stimulation at 14 days was associated with both an elevation in opsonin levels and an associated hepatic and splenic enlargement. The decline in phagocytic activity over the 14-30 day interval was associated with a progressive decline in the plasma opsonic activity, a return of the spleen to its normal size in relationship to the body weight, and a persistent hepatomegaly. These findings suggest that the alterations in macrophage function during tumour growth may be mediated in part by changes in the opsonic or phagocytosis promoting capacity of plasma. Since opsonic protein contributes to the discriminatory capacity of macrophages, it is suggested that changes in the blood opsonin level may condition the anti-tumour capacity of the macrophage system with respect to host defence aginst malignant disease.


The anti-tumour activity of methyl-beta-cyclodextrin (MEBCD), a cyclic oligosaccharide known for its interaction with the plasma membrane, was investigated in vitro and in vivo and compared with that of doxorubicin (DOX) in the human tumour models MCF7 breast carcinoma and A2780 ovarian carcinoma. In vitro proliferation was assessed using the MTT assay. In vivo studies were carried out using xenografted Swiss nude mice injected weekly i.p. with MEBCD at 300 or 800 mg kg(-1) or DOX at 2 mg kg(-1), during 2 months. Under these conditions, MEBCD was active against MCF7 and A2780 cell lines and tumour xenografts. For each tumour model, the tumoral volume of the xenografted mice treated with MEBCD was at least twofold reduced compared with the control group. In the MCF7 model, MEBCD (800 mg kg(-1)) was more active than DOX (2 mg kg(-1)). After 56 days of treatment with MEBCD, no toxicologically meaningful differences were observed in macroscopic and microscopic parameters compared with controls. The accumulation of MEBCD in normal and tumour tissues was also assessed using a chromatographic method. Results indicated that after a single injection of MEBCD, tumour, liver and kidneys accumulated the highest concentrations of MEBCD. These results provided a basis for the potential therapeutic application of MEBCD in cancer therapy.


The 19F MRS measurements were done on mice bearing the human colon tumour xenograft (HCT116). The intratumoural conversion is greater when TAPET-CD/5-FC is delivered intratumourally (i.tu.) than when TAPET-CD is delivered intravenously (i.v.) and 5-FC intraperitoneally (i.p.). Repeat measurements of the same tumour also yielded important information on the tumour colonization by TAPET-CD through the correlated 5-FC to 5-FU conversion efficacy. The in vivo MRS spectra were confirmed by in vitro19F MRS of perchloric acid extracts of the tumour tissue. No 5-FU metabolites were detectable in vivo in the tumours. However, the in vitro measurements revealed, besides 5-FC and 5-FU, the presence of small amounts of catabolites. Finally, spectra obtained in vitro from liver extracts of tumour-bearing mice treated i.tu. with TAPET-CD/5-FC showed no 5-FU and only little amounts of catabolites. Our data illustrate most importantly the potential of 19F MRS to monitor biologically-based treatments involving cytosine deaminase.The aim of this study was to evaluate the applicability of fluorine-19 magnetic resonance spectroscopy ( New developments for cancer treatment are allowing individualized and biologically-based therapies. As part of these developments one needs to accurately define both physical and biological characteristics of tumours, and to monitor the efficacy of treatment on an individual basis. Techniques such as computed tomography (CT) or magnetic resonance imaging (MRI) have been shown extremely useful in defining the physical limits of the tumour. Progress has also been made with regards to the evaluation of biological parameters of the tumour microenvironment  and dynamic monitoring of therapy-induced changes  chemotherapy). Yet, further progress is of crucial importance for the evaluation of new biologically-based therapies that may be more tumour specific than traditional therapies, and which necessitate a stringent individualized follow-up. Technologies that are being explored in this context are positron emission tomography (PET) and magnetic resonance spectroscopy (MRS). The application of biological imaging methodologies is essentially non-invasive, and thus biopsies before and during the treatment do not need to be taken, leaving the tumour microenvironment undisturbed. Furthermore, these methods allow potential screening of tumours at many sites of the body and enable the direct comparison with normal tissues.19F) has high sensitivity (83% relative to 1H), and because only negligible amounts of 19F are normally present in the body. 5-FU is a well-known chemotherapeutic agent, and has been evaluated along with the various metabolites. Although 5-FU measurements are often done in plasma samples, studies indicate that plasma levels do not reflect the intratumoural activity of 5-FU , and to evaluate concentrations and pharmacology of fluorinated chemotherapeutics has been reported. Compounds such as 5-FU lend themselves to this methodology because fluorine-19   enzyme/prodrug system. The non-mammalian enzyme CDase converts the non-toxic 5-FC to the cytostatic drug (5-FU). Previous results showed that recombinant bacteria can be used as a delivery vector to transfer CDase selectively to the compromised intratumoural microenvironment. The tumour-selective applicability of this strategy has recently been demonstrated MRS has already been shown to be able to detect conversion of 5-FC to 5-FU in tumours in which cells have been engineered to express CDase or by using a monoclonal tumour targeting antibody-CDase conjugate  were investigated in a mouse-xenografted human tumour model.The objective of the present study is to demonstrate the applicability of 19F MRS.Our results show that the selective intratumoural production of 5-FU by a biologically-based therapy such as the TAPET-CD/5-FC strategy, can be monitored using s.c.) in nu/nu mice using 1 × 106 cells per 100 μl. The cells were inoculated into the lower flank. At the time of TAPET-CD injection and of 19F MRS, the tumour volumes were between 300 mm3 and 2000 mm3 .HCT116 human colon tumour was xenografted subcutaneous . The bacteria were grown in liquid modified LB medium at 37°C to a given number that was determined by OD600 nm measurements (see details in The bacteria used were attenuated i.tu.) injections of 1 × 108 to 3 × 108 bacteria per 100 μl were given in two directions which, considering the size of the tumour and the insertion depth of the needle, were mainly central. The TAPET-CD were injected at a very slow rate to avoid leakage of the suspension. The intravenous injections (tail vein) consisted of 5 × 105 to 1 × 106 bacteria. The time of bacterial injection was considered day 0.Intratumoural (n=7), 1 g samples were taken aseptically and randomly from tumours not used for in vitro MRS. The tissue samples were homogenized and suspended in 1 ml of saline. Bacterial colonization was subsequently quantified by serial dilution series in modified LB broth. All samples were kept at 37°C for 24 h, at which time the colony-forming units (cfu) per gram of tissue were determined.To evaluate the intratumoural colonization of the TAPET-CD  and centrifuged. The pellets were washed, sonicated, and the lysates were incubated at 37°C for 24 h with a 10 mg/ml solution of 5-FC to which Tris HCl (1 M) was added.The preparation of the bacterial lysates for 4 (1 M) per gram tissue. After homogenization, the sample was incubated on ice for 1 h followed by centrifugation for 15 min at 1000 g (0°C). The supernatant was decanted and neutralized with KOH (10 M) and KHCO3 (1 M). The resulting precipitate was discarded following a second centrifugation. The supernatant was subsequently freeze-dried and the lyophilizate was dissolved in 0.5 ml of a potassium phosphate buffer .Animals were sacrificed by cervical dislocation and immediately afterwards the tumour and liver were removed and snap frozen in liquid nitrogen. The whole tumour or liver was homogenized at 0°C in 5 ml of cold HClOIn vivo19F MRS experiments on mice bearing the HCT116 tumour were performed at 188 MHz in a Bruker Biospec , equipped with a horizontal 4.7 Tesla superconducting magnet with 30 cm bore, using a 10 mm transmit-receive surface coil. Prior to the measurement, the mice were anaesthetized with intraperitoneally (i.p.) injected sodium pentobarbital . After administration of the 5-FC ; SIGMA, Belgium), the mice were placed on a Perspex plate such that the tumour was positioned directly above the circular surface coil. Following shimming on the water proton signal, serial 19F NMR spectra were acquired every 13 min during 1 to 2.5 h =0.75 s, number of averages (NA)=1024, spectral width=27 kHz; acquisition size=2048 points; no proton decoupling). The signals were processed by zerofilling to 4096 points and exponential multiplication of 6 Hz.19F MRS measurements were performed at day 0 and day 1. Some animals were also evaluated 1 or 2 weeks after the injection of TAPET-CD. Before a new injection of 5-FC was given, a spectrum was first recorded to determine whether there were still signals present from the previous injection (24 h earlier).Most i.tu. injection of 5-FC (as before) and 5-FU  in tumour-bearing mice treated with TAPET-CD and were also correlated to the 1 H chemical shift of the tumour H2O resonance. A spherical external reference sample  containing 4-fluorobenzoic acid  in deuterated water saturated with chromium acetylacetonate (Cr(acac)3, ACROS, Belgium) was placed ∼9 mm under the coil to monitor stability of the MRS technique and coil load changes for the different mice/tumours under evaluation. Absolute concentrations were determined in a separate experiment using a cylindrical water phantom sample  containing 1.25 mM 5-FC, 2.5 mM 5-FU and 10 mM NaF.The chemical shift of the 5-FU resonance was set to 0 p.p.m. The 5-FC signal was observed around 1.2 p.p.m. The signal positions were verified with the in vivo19F MRS measurements, the body temperature of the mice was kept at 36°C with the use of warm air ventilation in the magnet bore.During the In vitro analysis of tissue extracts of TAPET-CD/5-FC-treated animals and in vitro analysis of lysates of TAPET-CD cultures were performed in a high resolution AMX 360 (8.4 Tesla) spectrometer . Specific settings for the tissue extracts (278 K) were, TR=2.5 s, NA=3072 or 12288, 1H WALTZ-16-decoupling during the 1 s acquisition; and for the lysates (295 K), TR=2.5 s, NA=256, no proton decoupling. The tissue extracts were measured at 278 K to exploit the substantially shorter relaxation time at low temperature   with a coaxial FBA reference sample insert.All results are expressed as averages ± SEM (with indication of the number of experiments). Linear regression analysis was performed where appropriate.In vitro19F MRS measurements of the conversion of 5-FC to 5-FU were initially performed in lysates from a series of TAPET-CD cultures to be used in the experiments. The conversion capacity increased as a function of TAPET-CD growth status (OD600) and was optimal when lysates were evaluated at the maximum of the growth phase (R2=0.945) and confirmed the quality of the TAPET-CD stock used for the in vivo experiments.19F MRS to evaluate in vivo non-invasively the conversion capacity of the TAPET-CD in the tumour microenvironment. The proof-of-principle was first demonstrated using HCT116 tumour-bearing mice and local i.tu. injection of both TAPET-CD and 5-FC. Figure 1in vivo19F MRS measurements performed 1 day after the TAPET-CD injection, starting immediately after the 5-FC injection. The 5-FC signal was maximal in the first acquisition that started within 10 min after the 5-FC administration , and declined slowly during the experimental time of up to 2 h. The 5-FU resonance peak became visible during the first 30 min of the measurement, increasing continuously thereafter as shown with the serial spectra. Both 5-FC and 5-FU resonances were measured with adequate spectral resolution. No clear catabolites or anabolites of 5-FU could be observed during the total experimental time.We subsequently assessed the applicability of i.tu. injection.Spectra recorded 24 h after the initial 5-FC to 5-FU conversion measurement, showed complete absence of fluorine signals. Conversion of 5-FC to 5-FU was however again detectable in these tumours following a repeat 5-FC i.tu. TAPET-CD injection. We hypothesized that this was the result of insufficient bacterial colonization in these tumours. We therefore repeated the 19F MRS analysis at a later time point without any additional administration of TAPET-CD. A representative example of such a measurement is illustrated in Figure 2A19F MRS analysis was started within 10 min after the i.tu. 5-FC injection. n=6). These data highlight the importance of repeat measurements for detecting the presence of TAPET-CD conversion efficacy during the tumour colonization period.Intratumoural conversion of 5-FC to 5-FU was however not always present at day 1 following the i.tu. application but also the systemic delivery of the enzyme/prodrug system. Intratumoural production of 5-FU following i.v. injection of TAPET-CD and i.p. injection of 5-FC (300 mg/kg) was measured in vivo in the HCT116 colon tumour . A clear conversion of 5-FC to 5-FU was seen with this systemic application of TAPET-CD/5-FC, although the 5-FU signals were less than with the i.tu. delivery route  of extract preparations from these tumours. Intratumoural concentrations of 5-FU were about 0.5±0.2 mM (n=6) for the i.tu. and 0.15±0.07 mM (n=7) for the systemically TAPET-CD plus 5-FC treated tumours respectively. A representative example of this analysis , i.p. 0.025±0.025 mM (n=5)) and in some cases also a very small anabolite signal between 3.5 and 5.5 p.p.m. (∼0.015 mM) , small catabolite signals of α-fluoroureido-β-propionic acid (FUPA) at −17.3 p.p.m. and α-fluoro-β-alanine (FBAL) at −18.6 p.p.m. were observed in the liver extracts ) and the systemic administration (0.09±0.02 mM (n=6)).It is obviously important to evaluate normal tissue in parallel with tumour tissue, as both ultimately determine the therapeutic window of treatment. Besides the perchloric acid extracts of tumours, also extracts of snap-frozen livers were thus analysed with 19F MRS study we demonstrate the non-invasive detection of the dynamic production of 5-FU from 5-FC in the human HCT116 colon tumour xenograft following the use of cytosine deaminase- (CDase-) recombinant S. Typhimurium (TAPET-CD). The results show intratumoural conversion of 5-FC to 5-FU within 30 min which proceeded throughout the MRS measurement time extending up to 1.5–2 h after the 5-FC injection. This suggests that continued intratumoural production of 5-FU is greater than the diffusion of 5-FU from the tumour into the systemic circulation of the animal. The high resolution in vitro19F MRS analysis of the snap-frozen whole tumours confirmed the in vivo results. At the end of the MRS measurement, the i.tu. 5-FU signal corresponded to about 0.5±0.2 mM.In the present i.v. TAPET-CD and i.p. 5-FC, we measured an intratumoural 5-FU concentration of about 0.15±0.07 mM two hours post 5-FC injection. Although this is more than three times less than what is found after an i.tu. injection (0.5±0.2 mM), this still represents about 20 μg/ml 5-FU (based on 300 mg/kg 5-FC given i.p. in the present experiments). Lambin and colleagues discussed the potential of the bacteria-based CDase/5-FC strategy to improve the therapeutic outcome from the combination with radiotherapy .The difference in spectral quality for mice treated with 19F MRS measurements allowed us to gain a better understanding of some of the characteristics of the TAPET-CD/5-FC treatment strategy. First, a period ranging from 1 to 7 days seems necessary for the bacteria to establish and proliferate selectively in the tumours and express sufficient levels of CDase. This agrees with our previous work using the TAPET-CD/5-FC, in which the rat rhabdomyosarcoma tumour model was used  is being tested in clinical phase I studies (The present results are to our knowledge the first demonstration of the applicability of


Changes in cell mediated and humoral immunity following the excision of a transplantable melanoma growing in the footpad of its syngeneic host, as measured by an in vitro cytotoxicity test, were assessed. Spleen cell cytotoxicity did not change significantly. Cells from the regional lymph nodes stimulated tumour growth before tumour excision. Three days following tumour excision this stimulatory effect was undetectable. Loss of serum factors capable of blocking the cytotoxicity of spleen cells occurred 24 h after tumour excision. Serum cytotoxicity increased after tumour excision to a maximum of the third day. Following tumour excision the rise in serum cytotoxicity and loss of regional lymph node tumour stimulation were concomitant with the loss of blocking activity.


Leucocytes are a major component of the tumour microenvironment. Recent studies have indicated that the infiltration and activity of these host cells are regulated by the tumour to promote its survival and progression. Through the production of an array of growth factors, proteases and angiogenic mediators, leucocytes in the tumour microenvironment promote tumour growth, angiogenesis and metastasis. However, controversy remains over the relationship between these host cells and tumour progression. In the past, their presence has been construed as evidence for a host response against the growing tumour. This is because such immune functions were often observed in transplantable tumour models that by their nature represented a transplant that could elicit immune rejection despite being placed in immunocompromised hosts. However, it is becoming clear that tumours growing naturally are largely recognized as self and lack strong foreign antigens. Instead, they appear to have been selected to manipulate the host immune system to prevent rejection . This la culture .Under normal physiological circumstances, leucocytes are recruited in response to wounding, inflammatory or pathogenic stimuli. They are attracted by the local synthesis of chemokines (chemoattractive cytokines), cytokines and growth factors as well as products of tissue breakdown. These are all part of a signalling system that involves recognition of the pathological state, organisation of an appropriate cellular response and suppression of this response once the situation is resolved. During cutaneous wound healing, these processes require epithelial cell proliferation and migration, angiogenesis and tissue remodelling . In tumoSeveral steps are crucial for a tumour to become metastatic. Tumour cells need to be able to break out of their confining basement membranes in order to enter the extracellular matrix and circulation. These processes require the proteolytic breakdown of basement membranes, changes in epithelial cell adhesion, migration and the suppression of ankoisis. They are matched in the surrounding stroma with angiogenesis as well as the frequent recruitment of leucocytes. Angiogenesis, known to be the crucial process for tumour progression by providing oxygen and nutrients and removal of waste products, as well as, providing an expanding endothelial surface for the tumour cells to enter the circulation, also involves degradation of basement membranes followed by migration of endothelial cells into the tumour stroma . Recent Macrophages derived from circulating monocytes represent a major component of the infiltrated leucocytic population in the tumour microenvironment. These cells have a wide range of functions in immunity, during development and in tissue repair. They can adopt a particular phenotype according to the demand and produce many factors ranging from chemokines, cytokines, and proteases, to angiogenic and growth factors. They therefore appear to be the ‘jack-of all trades’ of the myeloid lineage. Of all the cells of the myeloid lineage, the evidence is strongest in support of a positive impact of macrophages on tumour progression. For example, in greater than 80% of clinical studies, an increase in tumour-associated macrophages (TAMs) density is correlated with poor prognosis, with less than 10% of studies showing the converse . One sucSeveral experiments have supported the role of macrophages in tumour progression. We have observed that in a Polyoma Middle T antigen-induced mouse model of breast cancer (PyMT), an increase of macrophage infiltration at the primary tumour site occurred immediately before the onset of malignant transition  and Thymidine Phosphorylase (TP) (β (TGFβ), platelet-derived growth factor, interleukin-6 (IL-6), urokinase plasminogen activator and Tissue-type Plasminogen Activator (t-PA) that may cause degradation of extracellular matrix to facilitate the tumour cell invasion and migration and induce angiogenesis  macrophages used to promote tumour progression are still unknown; however, it has been proposed that macrophages may promote tumour growth and angiogenesis through the production of growth factors and angiogenic inducers such as Epidermal Growth Factor (EGF), vascular endothelial growth factor (VEGF), tumour necrosis factor (TNFase (TP) . Macrophogenesis . Moreoveogenesis .β and prostaglandin E2 (PGE2), and TAM-produced factors such as IL-10 and PGE2, contribute to the general immunosuppression of the host as well as the antitumour activity of macrophages  also play an important role in both the activation of antigen-specific immunity and the maintenance of tolerance, providing a link between innate and adaptive immunity. Several clinical studies have reported the presence of DC within human tumours such as the stomach, colon, prostate, kidney, thyroid, breast and melanoma . The relAn infiltration of mast cells has been found in a variety of human cancers, including non-small-cell lung cancer , whereasα , CCL-2, Macrophage Inflammatory Protein MIP-1α and β, many of which can contribute to the tumour microenvironment by enhancing tumour cell growth and invasion either directly or through intermediaries such as macrophages  may also directly stimulate tumour proliferation in melanoma (Solid tumours are not just composed of malignant cells, but are complex microcosms of many cell types including a wide range of haematopoietic cells. The evidence described above suggests that cells of the myeloid lineages, particularly macrophages, mast cells and neutrophils, on balance play an active role in enhancing tumour progression and metastatic capacity. This is through their ability to promote angiogenesis and tissue remodelling as well as direct effects on epithelial cell viability, growth and migration. In wound healing or in response to an inflammatory stimulus, a similar panoply of cells is recruited. Sentinel cells, particularly macrophages and mast cells that send out chemotactic signals that in the first wave, bringing in neutrophils and monocytes, initiate this recruitment. These not only eliminate pathogens but also effect tissue repair, a process that involves angiogenesis and an induction of vascular permeability, tissue remodelling and the migration and proliferation of epithelial cells. These events are coordinated by a sophisticated and as yet, not understood language of soluble mediators involving cytokines, chemokines and growth factors.Similarly, during development, myeloid cells, particularly macrophages, play an important role in tissue formation and their absence often results in attenuated poorly formed structures in tissues as wide ranging as bone, skin and mammary gland . It woulThe tumour microenvironment also educates those invading cells to promote epithelial growth, viability, motility and invasion. Thus, it is noticeable in many tumour types that there are dense lymphocytic infiltration sites adjacent to areas of basement membrane breakdown and tumour invasion . In our Ablation of macrophage recruitment in transplantable breast cancers by a chemokine receptor antagonist significantly inhibited tumour development (


Stroke volume variation (SVV) has repeatedly been shown to be a reliable predictor of fluid responsiveness. Various devices allow automated clinical assessment of SVV. The aim of the present study was to compare prediction of fluid responsiveness using SVV, as determined by the FloTrac™/Vigileo™ system and the PiCCOplus™ system.FloTrac was determined via radial FloTrac sensor, and SVVPiCCO and pulse pressure variation were assessed via a femoral PiCCO catheter. Stroke volume was assessed by transpulmonary thermodilution. All variables were recorded before and after a volume shift induced by a change in body positioning (from 30° head-up position to 30° head-down position). Pearson correlation, t-test, and Bland-Altman analysis were performed. Area under the curve was determined by plotting receiver operating characteristic curves for changes in stroke volume in excess of 25%. P < 0.05 was considered statistically significant.In patients who had undergone elective cardiac surgery, SVVFloTrac and SVVPiCCO decreased significantly. Correlations of SVVFloTrac and SVVPiCCO with change in stroke volume were similar. There was no significant difference between the areas under the curve for SVVFloTrac and SVVPiCCO; the optimal threshold values given by the receiver operating characteristic curves were 9.6% for SVVFloTrac (sensitivity 91% and specificity 83%) and 12.1% for SVVPiCCO (sensitivity 87% and specificity 76%). There was a clinically acceptable agreement and strong correlation between SVVFloTrac and SVVPiCCO.Body positioning resulted in a significant increase in stroke volume; SVVPiCCO, SVVFloTrac has a lower threshold value.SVVs assessed using the FloTrac™/Vigileo™ and the PiCCOplus™ systems exhibited similar performances in terms of predicting fluid responsiveness. In comparison with SVV Fluid administration in critically ill patients is typically performed to increase cardiac preload, followed by a raise in cardiac output. However, studies conducted during the past few years have shown that about 50% of critically ill patients do not exhibit the desired effect (they are not fluid responsive) . Thus, wAs an alternative to these static variables, a dynamic approach may be used in the form of preload monitoring to guide fluid therapy. With passive leg rising in spontaneously breathing patients, the heart's reaction (increased venous return) can be assessed without any fluid administration , and in plus™ system  has repeatedly been shown to predict fluid responsiveness well in various clinical settings curve) for a SV increase greater than 25%. Threshold values for SVVFloTrac, SVVPiCCO and PPV were determined by considering values that yielded the greatest sensitivity and specificity. Based on these threshold values, the positive and negative predictive values for all dynamic variables were calculated. Additionally, regression analysis was performed for preload variables and SV changes. Comparison of SVVFloTrac with SVVPiCCO was done by Bland-Altman analysis and Pearson's correlation. ROC curves were compared in accordance with the method established by Hanley and coworkers [FloTrac and SVVPiCCO was compared by χ2 test. P < 0.05 was considered to be statistically significant. Unless otherwise stated, data are presented as mean ± standard deviation.All haemodynamic variables were recorded as mean values of three repeated measurements. Statistical analysis was performed using Statviewoworkers . Linear 40 patients  were enrolled in this study. During the study period, no significant changes in ventilatory tidal volumes or peak inspiratory pressure were identified.FloTrac, SVVPiCCO and PPV significantly decreased  and 12.1% for SVVPiCCO (sensitivity 87% and specificity 76%). Based on these threshold values, positive and negative predictive values were 80% and 92% for SVVFloTrac, respectively; corresponding values for SVVPiCCO were 77% and 90%. There was no significant difference between identification of patients with a SV increase of more than 25% using SVVFloTrac and SVVPiCCO (P = 0.523). Correlations between SVVFloTrac and SVVPiCCO and changes of SV (ΔSV) were similar  revealed a mean bias ± 2 standard deviations (limits of agreement) of -2.5 ± 6.1% for head-up measurements and -1.5 ± 3.6% for head-down measurements  were 0.612 and 0.757 for head-up and head-down measurements, respectively.Bland-Altman analysis  for SV increase greater than 25% was identified, whereas the SVV threshold value for the FloTrac/Vigileo™ system was 9.6%.Despite the clinical importance of SVV threshold values, little information is available in the literature. A SVV threshold value represents the 'trigger' value at which initiation of fluid replacement therapy is expected to result in a positive cardiac response. ROC analyses performed thus far have revealed that SVVstration . In a prstration  we obserplus system allows automated assessment of PPV. Reuter and coworkers [Flotrac and SVVPiCCO; it is likely that this finding is mainly accounted for by the study design. In this study preload changes were induced by changes in body positioning, whereas in the previous study [Variations in SV are followed by concomitant changes in arterial pressure, which can be assessed by measuring PPV. In addition to SVV, the PiCCOoworkers  found a oworkers , we demous study  a fluid us study .In contrast to SVV and PPV, conventional static preload parameters assessed in this study failed to predict fluid responsiveness, confirming the findings of previous work related to this issue ,23. CVP,PiCCO [PiCCO in a true fluid challenge [When interpreting of the data presented in this study, some methodological aspects and limitations must be considered. First, we induced a fluid shift by changing body positioning and not by subjecting patients to a 'real' fluid challenge. Changes in body positioning might have resulted in alterations to systemic vascular resistance based on sympathetic activity and not on changes in preload in isolation. Thus, a fluid shift due to changed body positioning and a true episode of hypovolaemia followed by intravenous fluid administration are not necessarily comparable. However, the sympathetic effect was minimized by using adequate sedation and analgesia in all patients during the study. Furthermore, the significant increase in GEDV observed after changed body positioning indicates an increase in preload. Moreover, body positioning is a typical manoeuvre for assessing fluid responsiveness ,6. In adPiCCO , and we hallenge . Second,FloTrac using intravenous fluid administration under different conditions and populations of critically ill patients is needed.Thus, our findings may not be directly applicable to other situations or to patients other than the population studied (patients with preserved left ventricular function after elective cardiac surgery). Therefore, further evaluation of SVVPiCCO, SVVFloTrac has a lower threshold value.In conclusion, SVV assessed using the FloTrac™/Vigileo™ and the PiCCOplus™ system exhibited comparable performance in terms of predicting fluid responsiveness. However, in comparison with SVV• Fluid responsiveness can reliably be assessed using SVV determined using the modified FloTrac™/Vigileo™ system.• SVV assessed using the modified FloTrac™/Vigileo™ and the PiCCOplus™ systems exhibited similar performance.FloTrac (9.6%) than for SVVPiCCO (12.1%).• The threshold value was lower for SVV• Compared with SVV, PPV was inferior in terms of predicting fluid responsiveness; static preload variables assessed in the study failed to provide valuable information on fluid responsiveness.FloTrac = SVV assessed using FloTrac™/Vigileo™; SVVPiCCO = SVV assessed using PiCCOplus™.AUC = area under the receiver operating characteristic curve; CVP = central venous pressure; GEDV = global end-diastolic volume; PPV = pulse pressure variation; ROC = receiver operating characteristic; SV = stroke volume; SVV = stroke volume variation; SVVThis study was supported in part by a research grant from Edwards Lifesciences . In the past, the Institute of Anaesthesiology and Intensive Care Medicine, Triemli City Hospital have held research grants from Pulsion Medical Systems  and Edwards Lifesciences. CKH has received lecture fees from both Pulsion Medical Systems and Edwards Lifesciences in the past.CKH was responsible for study design and protocol, patient recruitment, measurements, data collection, statistical analysis and manuscript writing. AS was responsible for patient recruitment, measurements, data collection and manuscript writing. LW was responsible for patient recruitment, data collection and technical support. AZ was responsible for study design and protocol.


A comparison has been made of the cachectic effects produced by the transplantable murine adenocarcinoma of the mouse colon (MAC16) with tumour necrosis factor-alpha (cachectin). Tumour necrosis factor-alpha  produced a dose-related weight reduction that was accompanied by a decrease in both food and water intake. The degree of weight loss was directly proportional to the decreased food and water intake. In contrast weight loss produced by the MAC16 tumour occurred without a reduction in fluid or nutrient intake. Both the MAC16 tumour and TNF-alpha produced hypoglycaemia and a reduction in the circulatory level of free fatty acids (FFA), but had opposite effects on the level of plasma triglycerides with the MAC16 tumour-induced cachexia causing a decrease and TNF-alpha producing an increase. The MAC16 tumour elaborated a lipolytic factor which caused an immediate release of FFA from adipose tissue. In contrast TNF-alpha had no effect on mobilization of adipose triglycerides over a short time period. Both TNF-alpha and extracts from the MAC16 tumour caused an enhanced release of amino acids from mouse diaphragm, which was suppressible with indomethacin and heat labile. No TNF was detected in the MAC16 tumour or in the serum of tumour-bearing animals. Both tumour and non-tumour-bearing animals responded with a similar elevation of their serum TNF levels 90 min after a single injection of endotoxin. It is concluded that weight loss produced by TNF-alpha arises from an anorexic effect and that this differs from the complex metabolic changes associated with cancer cachexia.


Several unique features of neuroblastoma (NB), including the capacity for spontaneous regression and maturation to benign pathology, suggest that genes that regulate cellular proliferation, survival and differentiation may be involved in directing clinical tumour aggressiveness. The in situ expression of Bcl-2, Rb, p21, p53 and Bax proteins, as well as the proliferation marker proliferating cell nuclear antigen (PCNA) were examined immunocytochemically in a selection of 38 stage- and outcome-identified NB tumours. Apoptotic cells were identified morphologically and by a DNA fragmentation labelling technique (TUNEL). Although the tumour cell density of Bcl-2, p53, Bax, PCNA and TUNEL positivity correlated with patient survival, a spatially organized expression pattern was further recognized in stroma-poor differentiating tumours. Immature tumour cells adjacent to thin fibrovascular stroma are proliferating, as evidenced by PCNA positivity, and often express Bcl-2. At increasing distance from this fibrovascular stroma, intermediately differentiated tumour cells express Rb, while with more advanced differentiation, proliferation ceases and Bcl-2 immunoreactivity is lost. The most differentiated tumour cells, which often express p53, and occasionally p21 and Bax, lie adjacent to TUNEL-positive, morphologically apoptotic cells. This spatial organization in favourable outcome NB tumours suggests that physiological regulation of differentiation and apoptosis may be involved in tumour regression.


MSC-10 tumour cells  were inoculated intramuscularly into thymectomized, X-irradiated isogeneic mice, either 48 h or 6 weeks after thymectomy and X-irradiation. Normal mice and immunologically reconstituted mice served as controls. A marked enhancement in frequency of tumour takes was observed in all groups of animals inoculated with tumour cells 48 h after whole:-body X-irradiation, whether thymectomized, immunologically reconstituted or not. The TD50 decreased to less than 1/10 of that observed in unirradiated controls. When mice were inoculated with tumour cells 6 weeks after X-irradiation, the incidence of tumour takes was similar to that of unirradiated controls, including the thymectomized-irradiated group, which was still severely immunodeficient as measured by antibody formation and skin graft rejection. The experiments indicate that whole-body X-irradiation creates a condition that favours tumour cell survival or growth. This "permissive state" exists only shortly after X-irradiation and is not correlated with the host's level of immunocompetence.


ProNPY processing of less than 50% was correlated with poor clinical outcome (P = 0.004). MYCN oncogene amplification was also correlated to a low degree of proNPY processing (P = 0.025). In summary, a low degree of proNPY processing was correlated to clinical advanced stage and poor outcome in neuroblastomas. ProNPY/NPY processing generated molecular forms of NPY with known differences in NPY-receptor selectivity, implicating a potential for in vivo modulation of NPY-like effects in tumour tissue. © 2000 Cancer Research CampaignNeuropeptide Y (NPY) is found at high concentrations in neural crest-derived tumours and has been implicated as a regulatory peptide in tumour growth and differentiation. Neuroblastomas, ganglioneuromas and phaeochromocytomas with significant concentrations of NPY-like immunoreactivity were investigated for different molecular forms of NPY and for significance of proNPY processing. Gel-permeation chromatography identified intact NPY (1–36) in all tumours, whereas proNPY (69 amino acids) was detected only in control adrenal tissue and malignant neuroblastomas. Purification of NPY-like immunoreactivity in tumour extracts and structural characterization revealed that both NPY (1–36) and the truncated form NPY (3–36) was present. The degree of processing of proNPY to NPY in tumour tissue was lower in advanced neuroblastomas with regional or metastatic spread (stage 3 and 4) (


Flow cytometry using the tumour perfusion probe Hoechst 33342 was employed to examine the distribution of photosensitisers in tumour cells located at different distances from the blood supply. Two tumour models, the SCCVII squamous cell carcinoma and FsaR fibrosarcoma growing in C3H/HeN mice, were used in the experiments. Among the photosensitisers tested, only BPD (benzoporphyrin derivative monoacid) exhibited uniform distribution in tumour cells irrespective of their distance from the vasculature. In this respect, 5-aminolaevulinic acid (i.e. its metabolite protoporphyrin IX), di- and tetrasulphonated aluminium phthalocyanines (A1PcS2 and AlPcS4), di- and tetrasulphonated tetraphenylporphines (TPPS2 and TPPS4), Photofrin and bacteriochlorophyll-a (i.e. its metabolite bacteriopheophytin-a) followed BPD in decreasing order in their efficacy of accumulation in tumour cells remote from the blood supply. This photosensitiser property appeared not to depend on tumour type, tumour size, route of photosensitiser administration, time after the administration, photosensitiser lipophilicity or on the presence of host cell infiltrate in the tumour. Following treatment with photodynamic therapy (PDT) in vivo, tumour cells were sorted based on their blood vessel proximity and their survival was determined by colony formation assay. The data demonstrate that the direct killing of tumour cells by Photofrin- and A1PcS2-based PDT decreases with increasing distance of the cells from the blood supply.


Vatsanabha  is a very well-known ingredient of Ayurvedic formulations and is prescribed as an antipyretic, analgesic, anti-rheumatic, appetizer and digestive. The recommended dose of purified Vatsanabha  root is 15 mg. We present a case of hypotension and bradycardia due to aconite poisoning caused by overdosing of an Ayurvedic medicine (Mahashankha Vati), which was primarily managed by Ayurvedic treatment.There are chances that the use of larger than recommended dose of Ayurvedic medicines containing aconite can produce drug reactions.  Ahiphena (Papaver somniferoum Linn.), Bhanga (Cannabis sativa Linn.), Dhattur (Dhatura metel Linn.), Karavira (Nerium indicum Mill.), Kupilu (Strychnos nuxvomica Linn.f.), Langali (Gloriosa superba Linn.), Vatsanabha , Jayapal (Croton tiglium Linn.), etc. have been used in Ayurveda medicine.[It is a common misconception among the public that Ayurvedic medicines are safe and devoid of adverse reaction. More than 70% of the total sales of Ayurvedic drugs are over-the-counter (OTC), thus leading to the use of Ayurvedic medicines without prescription, guidance and oversight of Ayurvedic physicians. Many poimedicine. Accordinmedicine. UnexpectVatsanabha  is used in Ayurveda as an antipyretic, analgesic, anti-rheumatic, appetizer and digestive.Aconite-based Ayurveda medicines are commonly used by Ayurvedic physicians and traditional practitioners/folk healers in primary healthcare. The most common aconite-based medicinal plant Aconite poisoning following use of herbal remedies has been reported from Hong Kong, India and Nepal.–6 ManageWe present here a case of hypotension and bradycardia due to overdose of aconite-based Ayurvedic medicine.A 47-year-old male reported to the OPD with the complaint of numbness around the mouth, nausea, heaviness of tongue, vertigo, black out, bursting type of pain over the back of the head, numbness and tingling sensation of palm and sole, and severe sweating which gradually reduced. The blood pressure was recorded as 30/10 mm Hg and pulse rate was 46 per minute with irregular rhythm, low volume and low tension. The extremities were cold and clammy, and there was retention of urine. There was no history of loss of consciousness or seizure. The patient was oriented and alert. The patient was a known case of hypertension but he had not taken antihypertensive drugs for the last 10 days. Five years ago, the patient had suffered from peptic ulcer syndrome.Mahashankha Vati along with two tablets of Citrakadi Vati without the advice of Ayurvedic physician or pharmacist of the institute, following complaints of abdominal pain and heaviness in the abdomen. A provisional diagnosis of aconite poisoning due to overdose of aconite containing Ayurvedic medicine (Mahashankha Vati) was made and the patient was admitted for observation.The patient had taken two tablets of Tankan bhasma (sodium biborate) and 1 g of Maricha Churna (Piper nigrum) was given orally with 50 ml of water. After 30 minutes, one glass of lukewarm milk with ½ tablespoon of ghee and 1 tablespoon of honey was given, followed by 20 ml of Arjunarista and 20 ml of Kanakasava with 40 ml of water. Thirty minutes later, the blood pressure was 60/30 mm Hg and pulse rate was 60 per minute with regular rhythm, low volume and low tension. Three hours later, the blood pressure and pulse remained the same and patient complained of severe fatigue and lassitude. Two bottles of 5% dextrose with normal saline (DNS) and one bottle of 10D (10% dextrose) along with 8 mg dexamethasone were given intravenously. Two hours later, the blood pressure was 100/60 mmHg and pulse was 70 per minute with regular rhythm, low volume, low tension. The patient had passed urine.Emesis was induced with salt water. Following this, 1 g of Arjunarista and 20 ml of Kanakasava were given with 40 ml of water along with 1 g of Tankan bhasma (sodium biborate) and 1 g of Maricha Churna (Piper nigrum) with 50 ml of water. Before these medicines were given, the patient had a light dinner of semi-liquid rice (Manda) and salt. The patient was discharged the next day after full recovery. He was prescribed 20 ml Dasamularista and 20 ml Punnavarista twice daily to be taken after meal with 40 ml of water to assist in the renal clearance of the aconite and to bring the tridosha into equilibrium state. Two capsules of Arjun (each capsule containing 250 mg of crude extract of Arjun) twice daily in the morning and evening after intake of food for 3 days.At this time, 20 ml of Vatsanabha  is a very well-known ingredient of Ayurvedic formulations and is prescribed as an antipyretic, analgesic, appetizer and a digestive. However, Vatsanabha root is always used after purification in these preparations. The process of purification involves submerging the roots into the fresh urine of a healthy cow for 3–7 days. Then, the outer covering of the root is pealed off and cut into small pieces and left in the sunlight for 1 day. After this, it is boiled with fresh cow milk for a minimum of 3 hours. Finally, it is dried in sunlight and powdered. The recommended dose of purified Vatsanabha  root is 15 mg.Tridosaja vikara, especially in Kapha vataj roga. Vatsanabha (A. ferox Linn.) is indicated in skin diseases, inflammatory diseases, anorexia, cough, bronchitis, fever, pain, rheumatic condition, and as an aphrodisiac also. The very popular classical Ayurvedic formulations containing Vatsanabha are Mrutunjaya rasa, Saubhgya vati, Rambana Rasa, Kaphaketu Rasa, Sanjeevani Vati, Moha Shankha Vati, etc.[Vatsanabha has significant therapeutic action in clinical and experimental studies.[samskaras” before being utilized in the Ayurvedic formulations. This study was undertaken in mice to ascertain whether “processed” aconite is less toxic as compared to the crude or unprocessed one. It was seen that crude aconite was significantly toxic to mice , whereas the fully processed aconite was absolutely nontoxic , and further, all the steps in the processing of aconite root were essential for complete detoxification.[Shodhana.[ati, etc. Vatsanab studies.10 Accordfication. PharmacoShodhana.13Vatsanabha contains 0.4–0.8% diterpene alkaloids and the concentration of aconite in the fresh plant is between 0.3% and 2.0% in tubers and 0.2% and 1.2% in the leaves. The highest concentration of aconite is found in the winter.[The tuber of e winter. The majoe winter. Aconitine winter.Aconite is a strong poison affecting several systems. Pure aconite can cause death at a dose of 2 mg, while 1 g of the aconite plant is fatal. Aconite toxicity is characterized by a burning or tingling sensation of the lips, tongue, mouth, and throat almost immediately after ingestion. Numbness of the throat and difficulty with speech may ensue. Nausea, vomiting, dizziness, fatigue, blurring of vision and paresthesia may also occur. Toxicity mainly affects CNS, heart and muscle tissues, primarily resulting in cardiovascular complications. Several case reports describe poisoning with aconite or its constituents, resulting in ventricular tachycardia, ventricular fibrillation, atrial fibrillation, and death. VentricuRecovery time is dependent on amount of intoxication; mildly intoxicated patients may take 1–2 days to recover, whereas patients with cardiovascular complications may take 7–9 days to recover.[Mahashankha Vati in which there are eight ingredients and Vatsanabha  is one of them. According to the Ayurvedic formulary of India and Bhaishajya Ratnavali, the recommended dose of Mahashankha Vati is 125–250 mg and it is indicated in grahani (irritable bowel syndrome), Arsha (piles), Mandagni (anorexia), etc.[Mahashankha Vati consumed by our patient (500 mg in weight) are shown in [vati therefore contains 17.8 mg of aconite.Our patient had consumed ia), etc. The consshown in . All theVatsanabha  by ingesting two tablets of Mahashankha Vati. There was a clear temporal relationship, the clinical features were diagnostic of aconite poisoning and there was no other clinical history to explain these features, making a diagnosis of overdose Vatsanabha  very likely. The patient was managed as per the treatment protocol of Visha chikitsa (poisoning treatment). Tankana bhasma (borax) is mentioned as an antidote of Vatsanabha in Ayurveda texts.[Maricha (P. nigrum) was added as it is a pramathi (has a purification effect). Arjuna  has significant positive inotropic properties in experimental models[Kanakasava contains atropine. As per Ayurveda, the properties of milk and ghee are opposite to those of poisons. Honey was added as Yogavahi . As there is no specific antidote for aconite overdosing, we gave symptomatic and supportive treatment,[Although the recommended dose of aconite is only 15 mg, our patient had consumed 70 mg of da texts. Maricha al models and Kanareatment, and the Vatsanabha  in a formulation and must use such drugs only in the recommended doses and under a physician’s supervision.The patient should be aware of the quantity of


The growth of s.c. Walker 256 carcinoma was found to be independent of secondary growths induced by i.v. injection. Tumour cells injected i.v. lodged mainly in the lungs, with small clusters of cells in the lymph nodes. The rate of cellular proliferation of these secondary growths of Walker carcinoma was significantly higher than that observed in the s.c. tumour. In addition, host lung tissue was found to inhibit the development of metastases, and it is postulated that the host tissue may produce a diffusible inhibitor and that differences in the effectiveness of these humoral factors may account, in part, for locational differences in tumour growth patterns.


An unusual primary adenomatoid tumour arising in the normal liver is described. Hepatectomy was performed, and the patient is alive and free of disease 1 year postsurgery. Grossly, the tumour showed a haemorrhagic cut surface with numerous microcystic structures. Histological examination revealed cystic or angiomatoid spaces of various sizes lined by cuboidal, low-columnar, or flattened epithelioid cells with vacuolated cytoplasm and round to oval nuclei. The epithelioid cells were entirely supported by proliferated capillaries and arteries together with collagenous stroma. Immunohistochemical studies showed that the epithelioid cells were strongly positive for a broad spectrum of cytokeratins  and mesothelial markers . These cells were negative for Hep par-1, carcinoembryonic antigen, neural cell adhesion molecule, CD34, CD31 and HMB45. Atypically, abundant capillaries were observed; however, the cystic proliferation of epithelioid cells with vacuoles and immunohistochemical profile of the epithelioid element were consistent with hepatic adenomatoid tumour. This is believed to be the first case report describing a solitary hepatic adenomatoid tumour.The hepatic adenomatoid tumour showed prominent cystic changes with an abundant vascular component, and immunohistochemical studies showed that it reacted with a broad spectrum of cytokeratins and mesothelial markers.When a hypervascular hepatic tumour is detected, adenomatoid tumour needs to be considered in the differential diagnosis.–Adenomatoid tumours are benign neoplasms of mesothelial origin and are most commonly encountered in the testis, epididymis, fallopian tube, uterus and ovary.1We report the case of hypervascular hepatic adenomatoid tumour incidentally discovered and diagnosed based on the findings of light microscopy and immunohistochemistry.A 39-year-old man without any remarkable previous medical history was introduced to our unit from another hospital for the treatment of a 2.0 cm hypervascular hepatic tumour in segment V; the tumour had been recently discovered by abdominal ultrasonography (US) performed during a general check-up. Physical examination was normal, and his carcinoembryonic antigen (CEA), carbohydrate antigen 19-9, α-fetoprotein, and protein induced by vitamin K absence-II levels were within the normal range. The patient was negative for both the hepatitis B virus surface antigen and the hepatitis C virus antibody.Abdominal US demonstrated a hyperechogenic round tumour in hepatic segment V. Abdominal CT demonstrated that the tumour was hypointense in pre-enhancement and highly enhanced in the arterial phase with early washout . In MRI,Grossly, the 1.5 cm soft mass in the subcapsular region showed a haemorrhagic cut surface with numerous microcystic structures and was non-encapsulated .Histologically, the well-circumscribed nodular tumour comprised cystic spaces of various sizes with collagenous stroma . The cysImmunohistochemically, the non-epithelioid cells were diffusely positive for α-smooth muscle actin (Sigma-Aldrich, St Louis, Missouri, USA; –We describe an unusual case of a hepatic adenomatoid tumour that was incidentally detected by radiography. Thorough clinical and radiological examinations did not reveal any evidence of another tumour. During preoperative diagnosis of the hypervascular hepatic mass, we suspected haemangioma or haemangioma-like tumour, epithelioid haemangioendothelioma and haemangiopericytoma, both of which occasionally demonstrate malignant characteristics. Hypervascular hepatic tumours such as hepatocellular carcinoma, haemangioma, focal nodular hyperplasia, hepatic adenoma and hepatic angiomyolipoma were also included in the differential diagnosis.8et al. reported that five adenomatoid tumours of the adrenal gland were strongly positive for CK7.4Grossly, the tumour resembled a haemangioma with regard to blood-related features and cystic, spongiotic appearance. However, the microscopic features of the tumour in our case were not consistent with those of any of the above-mentioned tumours. Neither hepatocellular proliferation nor simple endothelial proliferation was observed. This tumour was a soft, well-circumscribed, but non-capsulated mass with a prominently haemorrhagic and multicystic surface. Histologically, the tumour comprised cuboidal or flattened epithelioid cells arranged in a cystic or angiomatoid structure, supported by prominent capillaries and arteries. These epithelioid cells, which did not look like bile duct, were positive for a broad spectrum of cytokeratins such as AE1/AE3, CAM5.2, EMA, CK7 and CK19. Additional staining of our specimen for the mesothelial markers calretinin and D2-40 suggested the mesothelial origin of the epithelioid cells of the tumour. Isotalo Several common histological patterns have been observed in adenomatoid tumours, including adenomatoid, cystic, angiomatoid, tubular, plexiform and canalicular patterns. A combination of tubular and angiomatoid patterns is more commonly observed. Prominent cystic changes were observed in our case. The cystic type of adenomatoid tumour has been reported to arise in the uterus,An abundant vascular component has rarely been observed in typical adenomatoid tumours, but a vascularised component is present in some ovarian adenomatoid tumours.Immunohistochemically, the epithelioid cells reacted with a broad spectrum of cytokeratins and calretinin. Based on the morphological features and immunohistochemical profile, we believe that the characteristics of the hypervascular liver tumour are consistent with those of an adenomatoid tumour. Therefore, when a small-sized hypervascular tumour located in the subcapsular region is detected, the diagnosis of adenomatoid tumour should be considered.


Primary tumour cells were collected from peritoneal malignant effusions of patients with ovarian cancer. Acid cell extracts-induced CTL specifically lysed in an HLA-A2-restricted manner HER-2/neu+ autologous primary tumour cells as well as HER-2/neu+ tumour cell lines. In addition, adoptive transfer of such CTL significantly prolonged the survival of SCID mice xenografted with HLA-A2.1+, HER-2/neu+ human breast and ovarian tumour cell lines. Acid cell extracts collected from HLA-A2.1+ HER-2/neu negative (−) primary ovarian tumours induced HLA-A2.1-restricted CTL with weak in vitro and in vivo antitumour capacity, suggesting that HER-2/neu peptides within ACE from HER-2/neu-overexpressing primary ovarian tumour cells are immunodominant. The results presented herein serve as a rationale for the initiation of vaccination studies in patients with HER-2/neu-overexpressing ovarian tumours utilising autologous tumour-derived ACE.Unfractionated peptides (MW: up to 10 kDa), derived from HLA-A2.1 positive (+) HER-2/neu-overexpressing primary tumour cell acid cell extracts (ACE), were successfully used to generate  Tumour antigens, during the process of ‘crosspriming’ are taken up by antigen-presenting cells (APC) which present them to cytotoxic T lymphocytes (CTL), thereby eliciting the induction of antitumour immune responses  may be highly increased when tumour lysates are used as sources of antigenic material  . In addiIn vivo depletion studies in animal models indicated that most of this effect was mediated by CD8+ cells  with histologically confirmed ovarian cancer  were enrolled in this study. Patients fulfilled the following criteria: Karnofsky performance status >80%; bilirubin levels <1.7 ng dl−1 and creatinine levels <2.2 ng dl−1; leucocyte count >3.000 μl−1 and platelet count >100 000 μl−1. They had not received any antineoplastic therapy during the 3 week preceding the onset of the study. All patients were apprised of the study, and consents were obtained consistent with the policies of St Savas Cancer Hospital.HLA-A2.1g for 5 min to sediment cells. Malignant effusion-associated mononuclear cells (MEAMNC) and tumour cells were isolated from the cell pellet as previously described (+ cells) with almost equal numbers of CD3+CD8+ (28–37%) and CD3+CD4+ (32–40%) T cells. A substantial number of monocytes (CD14+ cells) was also detected (10–16%). In all cases examined, the number of MEAMNC isolated from the effusions ranged from 380 to 1.090 × 106 and that of tumour cells from 170 to 650 × 106. Both MEAMNC (as effectors) and tumour cells (as targets) were utilised in experiments when viability was >80%.Specimens of peritoneal effusions (1–2 l) from patients, collected during routine aspirations, were spun at 400 −1 rGM-CSF  and 500 U ml−1 rIL-4 , as described , comprising a mixture of CD3, CD7, CD19, CD45RA, CD56, and anti-IgE Abs coupled to MACS Microbeads according to the manufacturer's constructions. Dendritic cells were generated from monocytes in the presence of 800 U mlHER-2/neu expression was determined on single tumour cells isolated from the malignant effusions by flow cytometry, using the PE-conjugated anti-HER-2/neu mAb . The expression of HER-2/neu was qualified by comparing the mean fluorescence intensity (MFI) of the primary tumour cells with the MFI of tumour cell lines expressing HER-2/neu at different levels (i.e. HER-2/neu expression of the MDA-231 cell line is scored as 1 (negligible expression), of MCF-7 as 2 (intermediate expression) and of SKBR-3 as 3 (overexpression)) , followed by homogenisation in 1 ml homogenisation buffer. Eluates from cells were titrated with 10% trifluoroacetic acid and clarified by two successive centrifugations at 2500 g and 80 000 g for 30 min and 5 h, respectively. Peptides were processed immediately on a Sep-Pak C18 cartridge  equilibrated prior to use with 3 ml acetonitrile, followed by 3 ml deionised water. The eluate was allowed to flow through the cartridge by gravity, the column was washed with deionised water and bound material was finally eluted with 2 ml 60% acetonitrile in deionised water and lyophilised in a Speed-Vac . Dry product was reconstituted in HBSS and further processed on a Centricon centrifuge concentrator  with a cutoff of 10 kDa by centrifugation at 2500 g at 4°C for 2–3 h. The filtrate was aliquoted and stored at −20°C.This was performed as described (665), HER-2 (9689), HER-2 (9369), HER-2 (10952), HER-2 (9851) and HER-2 (9402). These are high binding affinity peptides for HLA-A2.1, eliciting strong CTL activity in vitro (154) and the MART-1/Melan-A-derived peptide Melan (927) were used as controls. The latter peptides have been demonstrated to elicit in vitro HLA-A2.1 CTL-restricted activity  using the Fmoc strategy and a 4-carboxybenzyl alcohol resin. Purification was performed by high-performance liquid chromatography. The purity was >95%. The following HER-2/neu-derived peptides were synthesised: HER-2/neu  or target cells (T2 cells) was determined from its capacity to induce maximal stabilisation of HLA-A2.1 expression on T2 cells (6 cells ml−1) were cultured in 24-well plates  with 1 × 105 ml−1 irradiated (3000 rad) autologous DC pulsed with ACE, derived from the autologous primary tumour cells, in a total volume of 2 ml X-VIVO 15 medium supplemented with 1% autologous serum (=complete medium), rIL-7 (20 ng ml)  and 25 IU ml−1 rIL-2  in CO2 incubators. After 5–7 days, one-half of the medium was replenished with fresh medium containing 40 ng ml−1 rIL-7 and 50 IU ml−1 rIL-2. After an additional 5 days incubation (=stimulation phase), recovered responders were washed and restimulated with thawed autologous irradiated (3000 rad) ACE-pulsed MEAMNC used as APC at a cell ratio of 1 : 2. Fresh rIL-2 (25 IU ml−1) was also added to the medium. After two additional rounds of restimulation, as above, (=restimulation phase) bulk MEAMNC effectors were tested in the in vitro cytotoxicity assays. Before transfer to SCID mice, the same effectors were expanded in tissue culture with anti-CD3 mAb  according to a method previously reported (Responder MEAMNC (1 × 106 ml−1) were placed in 100 μl aliquots into wells of 96-well V-bottomed plates (Costar). As targets, primary tumour cells or tumour cell lines were labelled with sodium [51Cr] chromate  and added to effectors at the indicated E : T ratios. For peptide recognition, T2 cells were incubated overnight at 26°C together with 20 μg ml−1 peptide (or ACE), washed and then labelled. Incubation was performed for 6 h in CO2 incubators. In some experiments, blocking with an anti-HLA-A2.1 mAb was performed by preincubating those target cells with 10 μg ml−1 of the BB7.2 mAb. Cytotoxicity values were considered to indicate significant recognition of a target when the differences between mean values  for percent lysis of the particular target  and unloaded T2 cells or HLA-A2− tumour targets were ⩾10% at an E : T ratio of 40 : 1 and statistically significant (P<0.05).The cytotoxicity assay was performed as previously described . Assays were performed according to the manufacturer's instructions.An ELISA kit specific for IL-2 was obtained from R&D Systems Europe. IFN-5 cells of each tumour cell line in 0.5 ml PBS. Injections with ACE-specific CTLs (2 × 107 cells in 0.5 ml PBS) were administered intraperitoneally (i.p.) at the time point when tumour was palpable (ca. 10–16 days after inoculation of mice with the tumour cell lines). Tumour size was monitored regularly every 4 days and was expressed as the product of the perpendicular diameters of individual tumours. Each animal experiment was repeated at least twice. The observation was terminated with the euthanasia of mice when the tumour mass grew up to 200–250 mm2 in diameter. A nonparametric Wilcoxon rank test was used in the statistical analysis of the size of the tumour in individual groups. The difference was considered statistically significant when P<0.05.Groups of 10 SCID mice were inoculated s.c. with 5 × 10+ primary tumour cells were tested for their ability to elicit specific CTL from autologous MEAMNC. These tumour cells were isolated from peritoneal effusions of three patients with ovarian cancer . Dendritic cells differentiated from monocytes within the MEAMNC population were used, pulsed with ACE, as APC. After a stimulation phase in the presence of IL-2 and IL-7, which was followed by three restimulations with autologous MEAMNC pulsed with ACE, as APC, the bulk effector MEAMNC population consisted of both CD8+ and CD4+ T cells . Although total MEAMNC effectors were utilised in the cytotoxicity experiments (see below), for simplicity reasons these are referred to as CTL. Bulk CLT cultures efficiently lysed T2 targets pulsed with ACE derived from the autologous ovarian primary tumour cells  and the HER-2/neu+ HLA-A2.1+ breast cancer cell line MCF-7  or the HER-2/neu− mouse fibrosarcoma cell line MC57X(H-2b). Cytotoxicity was HLA-A2.1-restricted since it was to a great extent inhibited in the presence of BB7.2 mAb (% range of inhibition against the autologous tumour and MCF-7 targets: 56–77 at a cell ratio of 40 : 1)  . The fin− HLA-A2.1+ Ova-4 and Ova-5 primary tumours induced only a modest CTL activity against the autologous primary tumour targets or the HER-2/neu+ HLA-A2.1+ SKOV3.A2 and MCF-7 tumour lines , with those in bulk cultures sensitised by ACE from the HER-2/neu+ primary tumours Ova-1, Ova-2 and Ova-3, thus excluding the possibility that low numbers of CD4+ T cells, apparently providing insufficient help for CD8+ CTL, could account for the low cytotoxic responses against the autologous primary tumour cells or the tumour cell lines.The relatively high percentages of cytotoxicity against the ACE-pulsed T2 targets + HLA-A2.1+ tumour targets with their ability to lyse T2 cells pulsed with HER-2/neu synthetic peptides known to represent HLA-A2.1-restricted CTL epitopes. As presented in 851), HER-2 (9435), HER-2 (9665) and HER-2 (9369). Peptides HER-2 (9435) and HER-2 (9369) were also recognised by CTL bulk cultures induced with ACE from Ova-2 and Ova-3 primary tumours. The same effectors lysed T2 cells pulsed with HER-2 (9689) but not the same targets pulsed with peptides HER-2 (9851) or HER-2 (9665). None of the effectors recognised HER-2 (10952) and HER-2 (9402) or control peptides Melan A (927) and gp (9154). In addition, there was no reactivity by any of the three CTL cultures against. As expected, CTL bulk cultures sensitised with ACE from the HER-2/neu− Ova-4 and Ova-5 tumours did not recognise any of the HER-2/neu peptides tested.To more precisely delineate the immunodominant role of HER-2/neu-derived peptides within the ACE from HER-2/neu-overexpressing ovarian primary tumour cells, we attempted to link the cytotoxic activity mediated by the ACE-induced CTL against the HER-2/neuγ and TNF-α were detected at varying concentrations. The data from γ and TNF-α levels produced by the MEAMNC bulk population sensitised with ACE from the HER-2/neu− Ova-4 and Ova-5 primary tumours  were almost within the range of concentrations quantitated in the supernatants of MEAMNC cultures stimulated by ACE from the HER-2/neu+ Ova-1, Ova-2 and Ova-3 primary tumours (range: 7.6–17.3 for IFN-γ and 1.3–10.5 for TNF-α). Only marginal levels of IL-4 could be detected in all five cases . These data demonstrated that the culture conditions used favour the production of Th1 cytokines supporting antitumour immunity. In addition, they support the idea that the low cytotoxicity induced by ACE preparations from Ova-4 and Ova-5 HER-2/neu− primary tumours should rather be attributed to an inadequate recognition of tumour targets than to qualitative differences in cytokine secretion profiles by the MEAMNC effectors.Parallel experiments were set up to quantitate cytokine production by the same ACE-induced MEAMNC effectors used in the cytotoxicity experiments. Cytokine levels were measured in culture supernantants by culture termination (i.e. after the third restimulation). With the use of commercially available ELISA kits, both IFN-in vivo by protecting SCID mice against the growth of the human tumour cell lines, which were previously used as targets in the in vitro cytotoxicity experiments. Cytotoxic T lymphocytes effectors after the restimulation phase were expanded in the presence of anti-CD3 mAb and rIL-2 and transferred at 2 × 107 cells per mouse to SCID mice with palpable subcutaneous tumours (these mice had been inoculated 10–16 days before with the tumour cell lines). As shown in 2 area) within 30–44 days in untreated animals. Such vigorous tumour growth was not restrained when mice were treated with a human CTL line specific for the HLA-A2.1-restricted epitope gp (9154) of the melanoma-associated gp100 protein -specific CTL). The same effectors remained without any effect when SCID mice were inoculated with the HER-2/neu+ HLA-A2.1− tumour lines SKBR3 or SKOV3 or with the murine MC57X fibrosarcoma , HER-2 (9435) , peptide HER-2 (9689), which may be recognised by Ova-2 and Ova-3 induced CTL, and peptides HER-2 (9851) and HER-2 (9665), which were recognised by one of the three bulk CTL. These HER-2/neu peptides have been demonstrated to bind to HLA-A2.1 molecules with high affinities  and the SKOV3 or SKBR3 tumour cell lines and therefore only HER-2/neu peptide-specific and HLA-A2.1-restricted CTL clones would have a chance to recognise them. Of course, recognition could also have been established through CTL clones specific for HLA-A2.1-restricted peptides other than those derived from the HER-2/neu oncoprotein. Although this is quite likely, still the contribution of such clones would not add much to overall killing of the HLA-A2.1+ tumour targets. This hypothesis is based on our findings demonstrating that CTL induced by ACE from the Ova-4 and Ova-5 HLA-A2.1+ but HER-2/neu− primary tumours only weakly lysed their autologous tumour targets (i.e. the Ova-4 and Ova-5 primary tumours) as well as the HLA-A2.1+ and HER-2/neu+ SKOV3.A2 and MCF-7 tumour cell lines. Thus, it seems possible that peptides within ACE from the HER-2/neu− primary ovarian tumours are weakly immunogenic most likely due to inefficient expression and presentation on the tumour cell surface. This is supported by the findings that both the Ova-4- and the Ova-5-ACE induced CTL, although exerting weak cytotoxic activity against the HLA-A2.1+ HER-2/neu− autologous primary tumours and against the HLA-A2.1+ HER-2/neu+ tumour cell lines, still were capable of (i) efficiently lysing T2 targets pulsed with the same ACE (i.e. those prepared from Ova-4 and Ova-5 primary tumours), and (ii) producing substantial amounts of IFN-γ and TNF-α after the restimulation phase. Both parameters were comparable to those of CTL induced by ACE from the HER-2/neu-overexpressing Ova-1, Ova-2 and Ova-3 primary tumours.Although HLA-A2.1 is the most popular allele for presenting HER-2/neu CTL epitopes, there are also other MHC class I alleles functioning as restriction elements for HER-2/neu peptide presentations , HER-2 (9435), HER-2 (9665) and HER-2 (9369)), Ova-2CTL (recognising HER-2 (9689), HER-2 (9435) and HER-2 (9369)) and Ova-3CTL (recognising HER-2 (9689), HER-2 (9435) and HER-2 (9369)) induced similar levels of protection demonstrating the immunodominance of these HER-2/neu epitopes, but not excluding, however, the possibility that also CTL clones specific for other HER-2/neu epitopes may be included in these bulk CTL populations.SCID mice were protected against inoculation with HER-2/neuin vitro the effector CTL population.There are several models described so far in the literature utilising different vehicles for vaccination studies with polyepitope constructs. These include attenuated virus vectors , our dat+ ovarian cancer. They also imply that vaccination with such multiepitope preparations may allow enhanced efficacy in the clinical treatment of ovarian cancer. Moreover, the identification of immunodominant HER-2/neu peptides within these unfractionated peptide extracts may help in the collection of peptides to be included in multipeptide vaccines.Collectively, the studies presented herein provide evidence that pooled peptides from HER-2/neu-overexpressing ovarian tumours can be utilised in cellular adoptive immunotherapy of patients with HER-2/neu


To date, the role of vascular endothelial growth factor (VEGF) in tumour growth has been attributed to the induction of angiogenesis. VEGF has been shown to be a survival factor for endothelial cells, preventing apoptosis by inducing Bcl-2 expression. In both murine (4T1) and human (MDA-MB-231) metastatic mammary carcinoma cell lines, we found that VEGF upregulated Bcl-2 expression and anti-VEGF antibodies reduced Bcl-2 expression. These alterations in Bcl-2 expression were reflected by the levels of tumour cell apoptosis. VEGF resulted in reduced tumour cell apoptosis, whereas its inhibition with anti-VEGF neutralizing antibodies induced apoptosis directly in tumour cells. Therefore, in addition to its role in angiogenesis and vessel permeability, VEGF acts as a survival factor for tumour cells, inducing Bcl-2 expression and inhibiting tumour cell apoptosis. © 2001 Cancer Research Campaign


A total of 152 non-small cell lung cancers (NSCLC) were studied retrospectively to determine the relationship between epidermal growth factor receptor (EGF-R) status and the histological type, tumour size, nodal status and prognosis. EGF-R status was assessed on routinely embedded paraffin sections with an antibody to the cytoplasmic domain of the tumour (F4 antibody). EGF was demonstrated in all tumour types and every squamous and large cell carcinoma was positive for the antibody. Most tumours showed heterogeneity of staining. EGF expression was seen statistically more frequently in well differentiated tumours. Patients with 50% or more tumour cells showing positivity tended to have an improved survival but this result failed to reach statistical significance. There was no relationship between the size of the primary tumour or the lymph node status. Other cells, such as mucinous glands, bronchial epithelial cells and macrophages stained positively with the monoclonal antibody. EGF receptor status, with the antibodies presently available, adds little to help in either diagnosis or prognosis. Interpretation of data has to be guarded since the antibody was seen in some normal cells.