A comparison of this information tothe Ly5b allele sequence identifies 12 allele-specific nucleotide changes. These basesubstitutions correspond to five amino-acid changes within the extracellular domain ofthe LCA molecule. These amino-acid differences are clustered in a region that alsocontains the greatest divergence between mouse and rat LCA sequences. Thus, thesetwo mouse LCA alleles exhibit a pattern of sequence conservation that mimics thatfound over a much broader scale of evolution. Analysis of antigenicity profiles for eachof the allelic sequence changes reveals three molecular domains of altered antigenicitythat could account for observed serological differences between the two alleles.Sequence information from the 5' end of the Ly5a LCA gene, generated usingpolymerase chain-reaction techniques on genomic DNA, reveals eight additionalnucleotide differences between the Ly5a and Ly5b alleles.The family of leucocyte common antigen (LCA) transmembrane glycoproteins isexpressed in most hematopoietic cells. Molecular isoforms of the LCA molecule aregenerated by alternative splicing of a single gene encoded on the murine chromosome 1.Three LCA alleles with different antigenic reactivities have been identified in inbredmouse strains. To investigate the divergence between alleles, cDNA clones to the SJA(Ly5


The terrain is well explored regarding genes whose gene expression is up-regulated upon binding of thyroid hormone (TH) to its nuclear receptor. This regulation mechanism has been intensively studied and is well understood. In contrast, a lot of white spots remain on the map when it comes to target genes whose expression is down-regulated upon binding of TH to the thyroid hormone receptor (TR). Since no consistent mechanism has been proposed to explain ligand-dependent down-regulation of target gene transcription several working hypotheses favour different molecular mechanisms. Some working theories suggest a direct binding of TR to regulatory elements of target genes. Others favour models that are independent of a direct DNA binding event. However recent data suggested that a direct binding of TR to DNA is dispensable for TH-dependent negative gene transcription. Thyroid hormone regulates gene expression in a positive and negative manner. The mechanism of positively TH-regulated gene transcription has been intensively studied and is well understood. The thyroid hormone receptor (TR) binds to thyroid hormone response elements (TREs) which are located within regulatory elements of TH target genes. If the ligand  is present, liganded TR recruits a huge coactivator complex. This coactivator complex possesses or recruits several enzymatic activities which modify the chromatin of target genes generating an open structure allowing for transcription. If the ligand is absent the un-liganded TR undergoes a conformational change which releases the coactivator complex and recruits a corepressor complex. Again this corepressor complex integrates several enzymatic activities modifying chromatin toward a closed and transcriptional silent state. These processes are well documented and discussed in comprehensive and excellent review articles elsewhere; see e.g. -3.Besides positively TH-regulated target genes there are clearly those genes whose gene expression is negatively regulated upon administration of TH. The probably best known example is the down-regulation of thyrotropin (TSH) in the pituitary as part of the negative endocrine hypothalamus-pituitary-thyroid feedback loop. However also in other tissues (e.g. the liver) negative gene regulation is a well known regulation pattern in microarray analysis -8. The p(A) TR binds to specific negative thyroid hormone response elements (negative TREs). Due to the specific composition of these DNA response elements the TR undergoes specific conformational changes leading to a recruitment of a coactivator complex in the absence of TH. In contrast, binding of the ligand recruits a corepressor complex to the liganded TR. Of note, the functional readout is diametrically opposed compared to the readout for positively regulated genes (see above).(B) A second model suggests that TR does not bind to DNA directly but rather to another transcription factor via protein-protein interactions. Again, the un-liganded TR (bound to DNA via transcription factor X) recruits a coactivator complex whereas the liganded TR recruits a corepressor complex.(C) Finally, a third model suggests that a soluble, not DNA-bound TR sequesters cofactors away from other DNA-bound transcription factor-cofactor complexes. Sequestering of corepressor components from DNA-bound transcription factors finally leads to an activation of gene expression whereas sequestering of coactivators leads to a repression of gene transcription.Several pros and cons exist for all of the three models and no uniform model is in sight which takes in account all aspects of negative gene regulation.Several reports described negative TREs Figure , model AOne major support for the model of negative TREs came from experiments by Frederic Wondisford and colleagues. They investigated the double mutant E125G; G126S within TRβ  which conserved the overall zinc finger structure of the DNA binding domain (DBD) but altered the DNA binding specificity . The GS If TR did not bind directly to DNA it might bind via protein-protein interactions to a transcription factor as a bridging factor in order to target TR to specific regulatory elements Figure , model BE457A knock-in mice harbouring a defective coactivator binding site demonstrated alterations in positively and – paradoxically – negatively regulated TH target genes [Following an alternative hypothesis TR might not bind to DNA at all (neither directly (model A) nor indirectly (model B)), but rather sequesters cofactors from DNA-bound transcription factor-cofactor complexes Figure , model Cet genes . On the et genes ,34. It rUp to now no convincing data have been presented doubtlessly supporting one particular model by discarding hitherto alternative models. Taking together recent data a direct binding of TR to negative TREs (model A) appears to be unlikely due to the lack of high-affinity binding sites for TR. Rather TR is involved in modulating the transcriptional activity of other transcription factors either directly (model B) or indirectly (model C). Clearly the DBD of TR participates in these processes independent of its function to bind DNA. Furthermore, cell-type and/or context-specific post-translational modifications might additionally contribute. Identification of TR binding sites by chromatin immunoprecipitation and correlation with adjacent DNA sequences (as successfully performed for the estrogen receptor ) are eagThe author declares that they have no competing interests.


Merkel cell carcinoma is an uncommon skin malignancy that has a high propensity for metastatic spread. A systematic literature search identified 17 cases describing metastasis to the gastrointestinal tract, with 7 cases involving the small or large bowel. To the best of our knowledge, this is the only case described of Merkel cell carcinoma metastasising to the ileocaecal valve.We present a 74-year-old Filipino woman diagnosed with Merkel cell carcinoma of the skin with regional node involvement. Following excision and radiotherapy, the tumour recurred with metastasis to the ileocaecal valve. The patient died 28 months after the initial diagnosis.The prognosis of metastatic Merkel cell carcinoma is poor. Currently the optimal management for metastatic disease is unclear and lacks a firm evidence base due to the small number of cases reported. Merkel cell carcinoma (MCC) is an uncommon and highly aggressive skin malignancy. It arises from Merkel cells at the dermo-epidermal junction, which are of neuroendocrine origin. Since its first description by Toker in 1972 .The incidence of MCC is 0.23 per 100,000 in Caucasians . At preset al. described the case of a 62-year-old man with axillary lymphadenopathy and metastasis to the stomach and the descending colon. The patient had a skin lesion excision which was initially diagnosed as basal cell carcinoma  OR "esophagus" [All Fields] OR "stomach" [All Fields] OR "duodenum" [All Fields] OR "jejunum" [All Fields] OR "ileum" [All Fields] OR "bowel" [All Fields] OR "intestine" [All Fields] OR "intestinal" [All Fields] OR "caecum" [All Fields] OR "rectum" [All Fields] OR "rectal" [All Fields] OR "colon" [All Fields] AND "merkel cell" [All Fields].This produced 61 results on PubMed of which 17 are reports of metastatic Merkel cell carcinoma in the gastrointestinal tract (excluding the liver).


Con A acceptor glycoproteins from the human Molt 4 (T cell leukaemia) and HeLa  cell lines were purified by affinity chromatography and used for the preparation of rat antisera. Cross-absorption analysis showed that each antiserum contained antibodies which recognised cell surface antigens preferentially expressed by the donor cell line. Molt 4-associated antigens were fully expressed on T cell tumour lines and normal thymocytes, but not on non T cell tumour lines, peripheral blood lymphocytes or other blood cells. Immunofluorescence studies showed that the antigens were preferentially expressed on a sub-population of immature thymocytes. HeLa-associated antigens were only fully expressed on one other epithelial tumour cell in a panel of 17 cell lines. Immunofluorescence studies showed that the HeLa-associated antigens were expressed on normal endocervical adenoepithelium but not on ectocervical, endometrial or intestinal epithelia. Thus purified Con A acceptor glycoproteins of cultured tumour cell lines are potent immunogens for the generation of antibodies recognising lineage-associated differentiation antigens. These antigens should be useful in tumour classification and in the study of normal differentiation.


The sampling rate of an injection every 3 min produces 20 results per hour from a flowing stream.An automated continuous monitoring system for the determination of inorganic and total mercury by flow-injection analysis followed by cold-vapour atomic absorption spectrometry is described. The method uses a typical flow-injection manifold where digestion and reduction of the injected sample takes place. Mercury is removed by aeration from the flowing stream in a specially designed air-liquid separator and swept into a silica cell for absorption measurement at a wavelength of 253.7 nm. A calibration curve up to 10 μg Hg ml


Xenopus of the MHCff genotype. The tumor cell can be transplanted in histocompatible larval ff hosts, but notin ff adults unless irradiated (3000 rad). The tumor is rejected by allogeneic hosts. Thetumor cells express neither markers of the B-cell lineage nor MHC encoded molecules;they express only markers of the T-cell lineage. Its lymphoid population is clonal asrevealed by the existence of a stable rearrangement pattern of the immunoglobulingenes. Cell lines growing continuously in vitro have been derived from the tumor.A spontaneous lymphoid thymus tumor was discovered in a male


The tumour specific transplantation antigen (TSTA) from a chemically induced rat sarcoma has been isolated as an electrophoretically homogeneous soluble material by affinity chromatography using a Sepharose bound antibody raised to the tumour in syngeneic rats. The TSTA is specific for the particular tumour used (the MC-1 sarcoma) and does not cross-react with material extracted from other rat sarcomata. In addition, a material with different physicochemical properties which cross-reacted with different sarcomata was also eluted from the antibody column and this may be the previously identified onco-embryonic antigen (OEA1) which is immunogenic in the syngeneic host.125I was used in a radioimmunoassay which detected soluble TSTA in rats bearing a MC-1 sarcoma. The assay shows that tumour transplantation is associated with a persisting release of soluble antigen into the circulation. This antigenic burden is present continuously and renewed as long as the tumour mass exists.The purified TSTA labelled with


An increasing number of renal dialysis-dependent patients with Arterio-Venous fistulae are undergoing cardiac surgery.The fistula has important effects on systemic hemodynamics in dialysis patients. The flow is significantly and positively related to cardiac output and cardiac index, and inversely related to pulmonary vascular resistance.Few problems are encountered on cardiopulmonary bypass despite left to right shunting of blood. We present an unusual case in which a large brachial Arterio-Venous fistula with large collaterals prevented weaning off cardiopulmonary bypass. Patients with chronic renal failure have a high prevalence of coronary artery disease and an increasing number are undergoing coronary artery bypass grafting. A-V fistula decreases mean systemic arterial pressure, effective systemic blood flow, total and pulmonary peripheral resistances, whereas it increases heart rate, total cardiac output, stroke volume, left atrial pressure, pulmonary arterial pressure ,2. In paIn these patients few problems are encountered on cardiopulmonary bypass despite left to right shunting of blood. We present an unusual case in which a large A-V fistula with large collaterals prevented weaning off cardiopulmonary bypass.2 and coronary angiogram showed significant two vessel disease involving the LAD and circumflex territory.A 77 yr old gentleman was admitted with an acute coronary syndrome with modest troponin rise. He was known to have aortic stenosis and coronary artery disease and the aortic gradient had accelerated by 50 mmHg over a 6 month period to 80 mmHg with good systolic but poor diastolic function. Valve area was calculated at 0.7 cmHe had End Stage Renal Disease for six years and was on chronic Haemodialysis via a surgical brachiocephalic Arterio-Venous fistula three times a week. The fistula was capable of flows averaging 400 ml/min during haemodialysis.Prior to surgery he became unstable and required an IABP necessitating urgent mechanical aortic valve and myocardial revascularization procedures.He had an uneventful anaesthetic induction and institution of cardiopulmonary bypass. During bypass it was noted that he required large doses of vasoconstrictors (pitressin infusion) to maintain perfusion pressure. He underwent a successful aortic valve replacement with a size 21 Tophat mechanical valve, reversed saphenous vein graft to obtuse marginal of circumflex and LIMA to LAD with a crossclamp time of 70 minutes.Detailed haemodynamic monitoring was performed from induction and cardiopulmonary bypass as depicted in figure.On attempting to wean him off bypass his diastolic pressure was very low which severely compromised coronary perfusion pressure and distended the Right ventricle. Three attempts were made to wean him off by-pass but all failed.P = 0.01) and systolic Pulmonary artery pressure .Temporary closure of the A-V access by a sphygmomanometer allowed successful weaning off bypass. The corresponding Pulmonary artery pressure and Cardiac Output values were compared before and after the A-V occlusion. Occlusion resulted in a transient decrease in cardiac output (from 11.4 ± 0.6 to 5.3 ± 0.5 l/min, Similar flow and pressure measurements were obtained on three separate occasions of closure and release of compression. Eventually the fistula was explored and tied off together with 3 large collateral vessels. Flow characteristics at baseline and with arterio-venous fistula occluded are shown in Figure The haemodynamic effects of arterio-venous fistulae are well known -3 but thIn practice most patients have distal radial-cephalic fistulae where the flow is lower than brachio-cephalic fistulae and these are successfully weaned off bypass without problems. However, as in this case proximal fistulas with extremely high flow rates can cause problems in combination with the effects of surgery and cardiopulmonary bypass on normal physiology including activation of complement and other plasma protein systems. This leads to low SVR and PVR which compounds the affect of the shunt. In this case the presence of three large collaterals linked to the fistula may have further augmented A-V access flow. These multiple interacting factors were clearly demonstrated and reproducible on temporary occlusion of the fistula and made weaning off cardiopulmonary bypass virtually impossible because of extremely low diastolic pressures leading to heart failure.Inadequate tissue perfusion and oxygenation was manifested by increased lactate production. The fistula may have contributed to this but other factors may have been responsible.This case serves to highlight a potential problem of cardiopulmonary bypass in patients with chronic large proximal A-V fistulae which have developed collaterals. In such cases it may be wise to measure fistula flow prior to surgery and place an uninflated sphygmomanometer in anticipation of temporary occlusion.


The work presented here has concerned the study of early, as well as late, enzymic changes occurring during diethylnitrosamine-induced hepatocarcinogenesis and liver regeneration after partial hepatectomy in comparison with normal liver differentiation. Rank correlation analysis of the enzyme data suggested a step-wise retrodifferentiation i.e. that the liver during carcinogenesis first assumed a neonatal enzymic pattern before attaining a foetal enzymic state. Similar enzymic changes were observed in regenerating liver after partial hepatectomy; again there was a step-wise retrodifferentiation of enzymic pattern and at 3 days post hepatectomy the liver had an enzymic pattern similar to both foetal and neoplastic liver. However, in contrast to liver undergoing neoplastic change, the regenerating liver retained the capacity to undergo redifferentiation towards a normal adult biochemical pattern.


Burkholderia pseudomallei was isolated from environmental specimens 1 year after an outbreak of acute melioidosis in a remote coastal community in northwestern Australia. B. pseudomallei was isolated from a water storage tank and from spray formed in a pH-raising aerator unit. Pulsed-field gel electrophoresis confirmed the aerator and storage tank isolates were identical to the outbreak strain, WKo97.


DCC - and c-erbB-2 genes in disseminated breast cancer cells and actuarial relapse-free survival. Furthermore, increasing numbers of genomic imbalances measured in disseminated tumour cells were significantly associated with worse prognosis of recurrent disease. Logistic regression and Cox multivariate analysis led to the identification of genomic imbalances as an independent prognostic factor. Determination of disseminated tumour cells by genotyping of oncogenes and tumour suppressor genes seems not only to be a useful adjunct in follow up of carcinoma patients but provides also valuable additional individualized prognostic and predictive information in breast cancer patients beyond the TNM system. © 2000 Cancer Research Campaign http://www.bjcancer.comThe prognostic value of disseminated tumour cells derived from 353 breast cancer patients was evaluated. Disseminated tumour cells were purified from blood using a newly established method and nucleic acids were subsequently isolated. We investigated genomic imbalances (GI) such as mutation, amplification and loss of heterozygosity of 13 tumour suppressor genes and 2 proto-oncogenes using DNA from isolated minimal residual cancer cells. Significant correlations were found between genomic alterations of the


The objectives of this study were to better our understanding of the carcinogenesis of gestational trophoblastic tumours and to investigate the possible presence of mutational alteration of the p53 tumour-suppressor gene in these tumours. Amplification-based direct DNA sequencing was performed on 14 hydatidiform moles, six invasive moles, eight choriocarcinomas and ten normal early placental tissues. No mutation in exons 5-8 was detected in any of these 38 tissue specimens. These results suggest that a mutation in p53 tumour suppressor either does not exist or is a very rare event in gestational trophoblastic tumours. The gestational trophoblastic tumours probably involve a tumour-suppressor gene other than p53 gene or may follow a completely different pathway to their malignant phenotype.


Doppler echocardiography is useful in the initial evaluation and long-term follow-up of patients with pulmonary artery hypertension. Aerosolised iloprost has been shown to reduce pulmonary pressure immediately after inhalation. We report the echocardiographic findings in a patient with severe pulmonary hypertension, before and after the inhalation of aerosolized iloprost. These findings illustrate the acute influence of iloprost in right and left ventricular hemodynamics and morphology. These findings were reproduced in subsequent echocardiographic evaluations. Doppler echocardiography is useful in the initial evaluation and long-term follow-up of patients with pulmonary arterial hypertension ,2.-1 with estimated right ventricular systolic pressure (RVSP) of 122 mmHg. She was then started on 5.0 μg of nebulized iloprost six times per day. At the 29th week of pregnancy Doppler echocardiography was repeated before and immediately after a single dose of 5 μg of iloprost. Postinhalation acute assessment revealed not only a remarkable reduction in RVSP but also an impressive change in cardiac morphology as is evident from figures A 21-year-old woman diagnosed with idiopathic pulmonary hypertension since she was eleven, responsive to calcium channel blockers, was referred for Cardiology consultation at 23 weeks pregnancy. She was on nifedipine and fraxiparine and Doppler echocardiography revealed a dilated and hypertrophied right ventricle and a peak tricuspid regurgitant jet velocity of 5.3 msThe findings that we describe showed the potential utility of echocardiography in the evaluation of the acute effects of drugs used in the treatment of pulmonary arterial hypertension.Two-dimensional parasternal view before iloprost inhalation. Two-dimensional parasternal long and short axis views before iloprost inhalation showing a small left ventricle with increased eccentricity index.Click here for fileTwo-dimensional parasternal view after iloprost inhalation. Two-dimensional parasternal long and short axis views after iloprost inhalation showing near normal morphology of both ventricles.Click here for fileThree-dimensional parasternal view before iloprost inhalation. Three-dimensional parasternal long axis view before iloprost inhalation showing a small left ventricle with increased eccentricity index.Click here for fileThree-dimensional parasternal view after iloprost inhalation. Three-dimensional parasternal long axis view after iloprost inhalation showing near normal morphology of both ventricles.Click here for file


A lymphocyte clonal assay developed to quantitate in vivo somatic cell mutations at the hypoxanthine-guanine phosphoribosyltransferase locus was modified in order to study resistance to methotrexate. Even though nucleoside-free culture conditions were used methotrexate was not lethal to lymphocytes plated into micro-wells at greater than 10(2) cells/well. HPLC analysis of supernatants from wells plated initially with 10(4) cells/well in 100 microM methotrexate revealed the presence of micro-molar levels of hypoxanthine and thymidine by the 5th and 8th day of culture respectively. When lymphocytes were plated at less than or equal to 10(2) cells/well in nucleoside free medium, methotrexate was cytotoxic and micro-molar levels of thymidine together with hypoxanthine protected lymphocytes cultured under these conditions from toxicity. Modulation of nucleic acid antimetabolite cytotoxicity by nucleosides and bases has been recognised for some years. Nucleoside free culture conditions have been advocated for studying cellular sensitivity to antifolates to avoid such interfering factors. However our results indicate that metabolites from dying or damaged cells can prevent methotrexate cytotoxicity, further complicating the development of a suitable clonogenic assay for investigating antifolate sensitivity.


The effect of methotrexate on solid rodent tumours has been investigated using the spontaneous mammary adenocarcinoma of the female C3H/Bts mouse. As these tumours exhibit a wide range of volume doubling times, calculations of tumour response to methotrexate must be related to doubling time. When methotrexate is injected into the tumour a dose-dependent tumour response is obtained. Systemic citrovorum "rescue" prevents methotrexate lethalities but reduces tumour response by a factor of 2. Methotrexate-treated tumours have an increased volume doubling time post treatment.


The effectiveness of cancer radiotherapy is compromised by the small proportion (approximately 5%) of patients who sustain severe normal tissue damage after standard radiotherapy treatments. Predictive tests are required to identify these highly radiosensitive cases. Patients with the rare, recessively inherited, cancer-prone syndrome ataxia-telangiectasia (A-T) sustain extremely severe normal tissue necrosis after radiotherapy and their cultured cells are also highly radiosensitive. Clinically normal carriers (heterozygotes) of the A-T gene have an increased risk of breast cancer, account for approximately 4% of all breast cancer cases and show a modest increase in cellular radiosensitivity in vitro. It has been suggested that a substantial proportion of highly radiosensitive (HR) breast cancer patients may be A-T heterozygotes, and that screening for mutations in the A-T gene could be used as a predictive test. We have tested this hypothesis in a group of cancer patients who showed adverse reactions to radiotherapy. Sixteen HR breast cancer patients showing mainly acute reactions (and seven HR patients with other cancers) were tested for ATM mutations using the restriction endonuclease fingerprinting assay. No mutations typical of those found in obligate A-T heterozygotes were detected. If the estimate that 4% of breast cancer cases are A-T gene carriers is correct, then ATM mutations do not confer clinical radiosensitivity. These early results suggest that screening for ATM mutations in cancer patients may not be of value in predicting adverse reactions.


TP53 mutation has been found, was not significantly different to that of normal strains. This was in contrast to LF strains that carry TP53 mutations. Cytogenetic observations of numerical and structural chromosome abnormalities were made on Giemsa stained metaphases prepared at different times during the lifespan of strains. Five strains from different LF families showed significantly increased frequencies of abnormal cells during the last 10% of their lifetime compared with seven normal strains and three other LF strains fell outside the normal range but did not reach significance. Two LF strains fell within the normal range indicating heterogeneity of the phenotype in this subset of LF fibroblasts. Numerical aberrations were the major aberration type observed. These observations of genetic instability are similar, but generally less strongly expressed, to those seen in LF strains with TP53 mutations. The basis for genetic instability in LF strains without TP53 mutations is not known, but appears not to involve defects in either the G 1 checkpoint or the checkpoint kinase hChk2. © 2000 Cancer Research CampaignThe mean in vitro lifespan of dermal fibroblast strains derived from cancer-affected individuals belonging to families conforming to the classical Li-Fraumeni-syndrome or the Li-Fraumeni-like syndrome (LF strains), but in whom no


A cone beam circular half-scan scheme is becoming an attractiveimaging method in cone beam CT since it improves the temporalresolution. Traditionally, the redundant data in the circularhalf-scan range is weighted by a central scanning plane-dependentweighting function; FDK algorithm is then applied on the weightedprojection data for reconstruction. However, this scheme stillsuffers the attenuation coefficient drop inherited with FDK whenthe cone angle becomes large. A new heuristic cone beamgeometry-dependent weighting scheme is proposed based on the ideathat there exists less redundancy for the projection data awayfrom the central scanning plane. The performance of FDKHSCW schemeis evaluated by comparing it to the FDK full-scan (FDKFS) schemeand the traditional FDK half-scan scheme with Parker's fan beamweighting function (FDKHSFW). Computer simulation is employed andconducted on a 3D Shepp-Logan phantom. The result illustrates acorrection of FDKHSCW to the attenuation coefficient drop in theoff-scanning plane associated with FDKFS and FDKHSFW whilemaintaining the same spatial resolution.


We describe an outbreak among drug users of severe soft-tissue infections caused by a clonal strain of group A streptococcus of M-type 25. Cases (n = 19) in drug users were defined as infections (mainly needle abscesses) due to the outbreak strain. Comparison with controls showed that infected drug users bought drugs more often at a specific place. Drug purchase and use habits may have contributed to this outbreak.


Tumour stromal neovascularization was investigated in 114 invasive and 20 in situ carcinomas of the uterine cervix by staining representative sections with the specific endothelial marker anti CD31 . A digital image analyser was used to measure the immunoreactivity. The following parameters were determined in the 'hot spots': vessel counts, vessel perimeter and endothelial stained area (expressed per mm2). The results were correlated with clinical and histopathological data. There was no significant relationship between the histopathological findings  and the median vessel count. In a univariate analysis all angiogenesis parameters had prognostic value: a higher vascularity was associated with worse prognosis (P < 0.05). Multiple regression analysis showed that vascular permeation (P < 0.001) and the median vessel count (P = 0.005) were the most important prognostic indicators. In the future these criteria may be used for selection of patients for anti-angiogenesis therapy.


Comparing the primary DCIS and the invasive or non-invasive recurrence, concordant histology was found in 62%, and identical marker expression in 63%. Although 11% of the recurrences developed at a distance from the primary DCIS, nearly all these showed the same histological and immunohistochemical profile. 5 patients developed well-differentiated DCIS or grade I invasive carcinoma after poorly differentiated DCIS. Although these recurrences occurred in the same quadrant as the primary DCIS, they may be considered as second primary tumours. Only 4 patients developed poorly differentiated DCIS or grade III invasive carcinoma after well differentiated DCIS. We conclude that in most cases the primary DCIS and its local recurrence are related histologically or by marker expression, suggesting that local recurrence usually reflects outgrowth of residual DCIS; progression of well differentiated DCIS towards poorly differentiated DCIS or grade III invasive carcinoma is a non-frequent event. © 2001 Cancer Research Campaign http://www.bjcancer.comWe have investigated primary ductal carcinomas in situ (DCIS) of the breast and their local recurrences after breast-conserving therapy (BCT) for histological characteristics and marker expression. Patients who were randomized in the EORTC trial 10853  and who developed a local recurrence were identified. Histology was reviewed for 116 cases; oestrogen and progesterone receptor status, and HER2/


Amplification and increased expression of many growth factor receptors, including the epidermal growth factor receptor (EGFR), has been observed in human tumours. One therapeutic strategy for overcoming EGF autocrine control of tumour growth is inhibition of EGFR protein tyrosine kinase (PTK). A series of low molecular weight molecules have been identified which inhibit the EGFR PTK in vitro and demonstrate antiproliferative activity against human cancer cell lines with high expression of EGFR. A significant growth delay in squamous cancer xenografts has been reported for one of these compounds, the tyrphostin RG13022. Based on these encouraging results, we sought to confirm the activity of RG13022 in vivo and relate the effects to the in vivo plasma disposition. RG13022 and three additional peaks were detected by HPLC following intraperitoneal administration of 20 mg kg-1 RG13022 in MF1 nu/nu mice. RG13022 demonstrated rapid biexponential elimination from plasma with a terminal half-life of 50.4 min. RG13022 plasma concentrations were less than 1 microM by 20 min post injection. A primary product was identified as the geometrical isomer (E)-RG13022. Both RG13022 and its geometrical isomer inhibited DNA synthesis in HN5 cells after a 24 h in vitro incubation (IC50 = 11 microM and 38 microM respectively). Neither RG13022 nor its geometrical isomer displayed significant cytotoxicity. RG13022 had no influence on the growth of HN5 tumours when administered chronically, starting either on the day of tumour inoculation or after establishment of tumour xenografts. The rapid in vivo elimination of RG13022 has potential significance to the development of this and other related tyrphostin tyrosine kinase inhibitors, as plasma concentrations fell below that required for in vitro activity by 20 min post injection. The lack of in vivo tumour growth delay suggests that a more optimal administration schedule for RG13022 would include more frequent injections or continuous administration. An improved formulation for RG13022 is therefore required before further development of this or other similar protein tyrosine kinase inhibitors can be made. Alternative strategies should also be sought which display longer lasting in vivo exposures.


We introduced the origin-defective SV40 early gene into cultured human oesophageal epithelial cells by infection of a recombinant SV40 adenovirus vector. The virus-infected cells formed colonies 3-4 weeks after infection in medium containing fetal calf serum. When the cells derived from 'serum-resistant' colonies were then maintained in the serum-free medium with a low calcium ion concentration, some of them passed the cell crisis and kept growing for over 12 months. These cells, regarded as immortalised cells, resembled the primarily cultured oesophageal epithelial cells in morphology and had some of their original characteristics. Treatment of the cells with a high calcium concentration induced phenotypic changes. These cells still responded to transforming growth factor beta. When the immortalised cells were injected into severe combined immunodeficient mice, they transiently formed epithelial cysts, although the typical differentiation pattern of the oesophageal epithelium was not observed. These cysts regressed within 2 months without development into tumours. The results indicated that human oesophageal epithelial cells were reproducibly immortalised by infection with a recombinant SV40 adenovirus vector at relatively high efficiency. The immortalised cells should be useful in studies on oesophageal carcinogenesis and in assessing the cooperative effects with other oncogene products or carcinogens.


Despite numerous studies in the past it is not possible yet to predict postoperative liver failureand safe limits for hepatectomy. In this study the following liver function tests ICG-ER(indocyaninegreen elimination rate), GEC  and MEGX-F(monoethylglycinexylidid formation) are examined with regard to loss of liver tissue andprediction of operative risk. Liver function tests were assessed in 20 patients prior to liverresection and on the 10th. postoperative day. Liver and tumor volume were measured byultrasound and pathologic specimen and the parenchymal resection rate was calculated. Inpatients without cirrhosis (n = 10) ICG-ER and MEGX-F remained unchanged afterresection, GEC was reduced but did not correspond to the resection rate. Patients withcirrhosis (n = 10) had a significantly lower ICG-ER and GEC before resection than patientswithout cirrhosis. After resection these tests were unchanged. Patients with liver relatedcomplications and cirrhosis (n = 5) had lower ICG-ER and GEC than patients with cirrhosisand no complications. In the postoperative course all liver function tests in these patients weresignificantly lower compared to preoperative results. Comparing liver function tests ICGserves best to indicate postoperative liver failure. Liver function tests do not correspond withloss of liver tissue.


Estimation of tumour proliferation may allow the design of individualised radiotherapy schedules to optimise response. This prospective study correlates the tumour proliferation rate of cervical carcinoma with response to conventional radiotherapy. The potential tumour cell doubling rate (Tpot) was estimated following flash labelling of the tumours in vivo using the DNA precursor, bromodeoxyuridine (BrdUrd); samples were analysed by flow cytometry. Tumour ploidy, DNA index and mitotic count were also assessed as was histological grade and type. Multiple biopsies from each tumour were obtained from 121 women. The median Tpot was 4.0 days, median S-phase duration 12.8 h and median adjusted labelling index 9.8%. Higher BrdUrd labelling was seen in patients who developed pelvic tumour recurrence following radiotherapy. This was the only biological/histological parameter with univariate and multivariate significance in relation to locoregional recurrence (P = 0.006 and P = 0.034 respectively). This study represents the first assessment of Tpot in relation to long-term response of cervical tumours treated by radiotherapy treatment. The association of high BrdUrd labelling and poor pelvic disease-free survival indicates the need for further research into the potential of radiotherapy schedule alteration to reflect tumour proliferation. The predictive value may be enhanced by combination with other biological parameters.


Objective: We sought to determine the effect of bacteria on fluorescence polarization (FPOL) testing of amniotic fluid.Methods:  Fusobacterium necrophorum and Escherichia coli were inoculated at concentrations of 103 and 106/ml in amniotic-fluid specimens from 4 patients with no clinical or laboratory evidence of infection. The FPOL results were obtained at inoculation and again at 24 h of incubation. The results were compared using analysis of variance (ANOVA).Results: The FPOL results from inoculated specimens were all within 2% of the uninoculated controls. The specimens incubated with bacteria showed a < 1–19% variation when compared with the time-zero uninoculated controls. However, uninoculated controls incubated for 24 h exhibited a 2–12% variation when compared with the time-zero controls, suggesting that the variation present was not secondary to the bacterial co-incubation.Conclusions: In vitro, neither bacterial inoculation nor prolonged co-incubation influences FPOL results beyond the effect of incubation alone. FPOL appears to be an appropriate test to assess fetal lung maturity in patients in whom intraamniotic infection is a concern.


Cellular repopulation in Lewis carcinoma irradiated with 60Co gamma-rays was examined by performing sequential cell-survival estimations using an in vitro soft-agar-colony assay. Following local irradiation (15--35 Gy) two distinct types of colony were seen: compact colonies with tightly packed cells and diffuse colonies with widely dispersed cells. Maximal diffuse colony formation in vitro was only obtained in the simultaneous presence of adequate numbers of compact colonies. After whole-body irradiation only compact colonies were observed. Only-cell survival data from compact colony counts correlated with cell survival estimated by the lung colony assay and we conclude that compact colonies are produced by clonogenic tumour cells. Cytochemical and immunological evidence showed that diffuse colonies were composed of macrophages. After local irradiation the initial kill of clonogenic tumour cells was dose dependent. At each dose level, repopulation began immediately and proceeded with a doubling time of about 1 day. Macrophage colony-forming cells (macrophage progenitors) per tumour were initially reduced by about 3 decades, but recovered very rapidly to reach pretreatment levels within 2 days. We conclude that at least two populations of clonogenic cells are present in Lewis lung carcinoma, tumour cells that repopulate irradiated tumours by in situ proliferation and host-macrophage progenitors that repopulate locally irradiated tumours by infiltration. The hazards of confusing host and tumour cell colonies in in vitro assay systems are stressed.


We report a patient with an implantable cardioverter defibrillator (ICD) for arrhythmogenic right ventricular dysplasia (ARVD) who received inappropriate shocks for atrioventricular node reentry tachycardia (AVRNT). Patient had multiple shocks for tachycardia with EGM characteristics of very short VA interval and CL of 300 msec. An electrophysiologic (EP) study reproducibly induced typical AVNRT with similar features. The slow AV nodal pathway ablation resolved the ICD shocks. Despite increasingly sophisticated discrimination algorithms available in modern ICDs, the ability to differentiate SVT from VT can be challenging. Our patient received inappropriate shocks for AVNRT. When device interrogation alone is not conclusive, an EP study may be necessary to determine the appropriate therapeutic course. Arrhythmogenic right ventricular dysplasia (ARVD) is an inherited degenerative disorder involving progressive replacement of the myocardium by fibro fatty tissue. This is seen predominantly in the right ventricle though left ventricular involvement is not uncommon. The fibrofatty replacement provides the substrate for life threatening arrhythmias such as monomorphic ventricular tachycardia, polymorphic ventricular tachycardia (torsades de pointes), ventricular fibrillation and / or electrical storm  are sophisticated devices with arrhythmia discrimination algorithms that reliably differentiate SVT from VT and provide appropriate therapy. We report a patient with ARVD who had inappropriate shocks for AVNRT.A 47-year old male presented with 3 ICD shocks for persistent ventricular tachycardia. He had known history of polysubstance abuse and coronary artery disease with angioplasty. Eight months prior to this admission he had presented with symptomatic monomorphic ventricular tachycardia (Cycle length 360 msec). A 2D-echo showed an ejection fraction of 40% followed by a coronary angiogram which showed non-obstructive disease with previously placed patent right coronary stent. Cardiac MRI confirmed a diagnosis of ARVD. He received a Biotronik Lexos DR ICD (model # 347000). Device interrogation during the current admission revealed a wide complex tachycardia (WCT) that received 3 ineffective shocks but resolved spontaneously. The WCT as shown by the Electrograms (EGMs) during ICD interrogation showed a short VA interval, a Cycle length (CL) of 300msec which were completely different from the VT seen during his initial clinical presentation . ICD shoDuring EP study, no arrhythmia was inducible at baseline. With RV stimulation and Isoproterenol (2mcg) provocation, a typical AVNRT with a pacing cycle length of 500 msecs and 3 extra stimuli of 290-280-270 ms, identical to the arrhythmia seen on ICD interrogation strips was easily and reproducibly induced . PresencOur patient presented with sustained monmorphic VT, was diagnosed as ARVD by cardiac MRI, requiring ICD implantation. Subsequently he had inappropriate shocks due to AVNRT . Due to differences in the characteristics of the two arrhythmias an appropriate diagnosis of the arrhythmia responsible for the inappropriate shocks could be established only with programmed electrical stimulation during an EP study.ARVD has an increased incidence of ventricular arrhythmias and SCD requiring ICD implantation. It has been estimated that about 20 percent of SCD among patients <35 years is due to ARVD .In spite of a high specificity, inappropriate shocks have been seen in ARVD patients with ICD especially with atrial arrhythmias. In a study of 653 arrhythmia episodes in patients with dual chamber ICDs, inappropriate detection of atrial arrhythmias (with a stable atrioventricular relationship) was seen in 42% of patients, of which atrial flutter or fibrillation were more often misdiagnosed [Most modern ICDs have sophisticated discrimination algorithms to differentiate atrial and ventricular arrhythmias. While ventricular arrhythmias are recognized and treated with precision, atrial arrhythmias can be misdiagnosed and receive inappropriate shock therapy. An EP study may thus be necessary in establishing an accurate diagnosis and delivering appropriate therapy.


Antibodies directed against the antigen released from viable tumour cells during growth have been raised by cross-immunization of immunocompetent hairy litter-mates with serum from nude mice bearing pancreatic tumour xenografts. Antisera raised against the components released from a primary pancreatic tumour xenograft (WB) or from a tumour cell-line xenograft (GER) showed a titre greater than 1:625 against cultured pancreatic tumour cells by an I125-binding assay. Five out of 14 of the hairy litter-mates immunized with serum from the same tumour (GER) produced antisera that bound more strongly to pancreatic cancer cells than to 13 other non-pancreatic cell lines (binding ratio greater than 2). Absorption of the antisera with pure CEA reduced the level of binding by 11-25% without affecting the specificity for pancreatic tumour cells. The antibody response could be completely removed by absorption with pancreatic tumour cells, whereas 50% and 18% of the activity remained after absorption with normal pancreas homogenate and a mixed non-pancreatic tumour-cell pool, respectively. Immunofluorescent staining of pancreatic tumour sections indicated that the antibody was localized on the membrane of ductular epithelial cells. Challenge of immunocompetent mice using this procedure has produced a polyspecific antiserum with signs of selectivity for the circulating antigens released from pancreatic cancer cells, and may provide a route to the production of antibody against specific tumour components.


Patients with neurogenic bladder due to spina bifida are considered to be at increased risk for aggressive bladder cancer. We present a unique case of a 32-year-old woman with spina bifida diagnosed with sarcomatoid urothelial carcinoma of the bladder and report diagnosis and management.A 32 year old woman with neurogenic bladder managed with intermittent self catheterisations, presented with gross hematuria. On cystoscopy, she had a bulky bladder mass on the posterior bladder wall. Bladder biopsies revealed sarcomatoid variant of bladder transitional cell carcinoma. Treatment included radical cystectomy with ileal conduit diversion and adjuvant chemotherapy with excellent intermediate term follow up.Patents with neurogenic bladder managed with intermittent self catheterisations need periodical follow up due to increased risk for aggressive bladder cancer. Immediate radical cystectomy with adjuvant chemotherapy is the suggested treatment approach. Patients with neurogenic bladder due to spina bifida (SB) are considered to be at increased risk for bladder cancer. These tumours are composed of urothelial, glandular or small cell component showing variable degrees of differentiation, while a small subset may have a prominent myxoid stroma . We repoA 32-year-old Greek white woman with neurogenic bladder due to spina bifida managed with intermittent self catheterizations since puberty; presented with gross hematuria and recurrent urinary tract infections. There was no history of bladder cancer in patient's family. Cystoscopy showed a bulky solid mass occupying the posterior bladder wall. Computed tomography didn't show nodal or distant metastases. Bladder biopsies revealed muscle-invasive sarcomatoid urothelial carcinoma with carcinoma in situ of the bladder neck. Radical cystectomy with anterior pelvic exenteration and ileal conduit formation was performed. Microscopically, the tumor was a high-grade urothelial carcinoma, Grade III according to the classification of the World Health Organization (WHO) of 1973, invading the whole bladder-wall and the pericystic lipoid tissue (stage pT3a). A large subset of the tumor assumed a spindle cell/sarcomatoid appearance with high mitotic rate and atypical mitoses. Immunohistochemically, the tumor cells were positive for the epithelial membrane antigen (EMA), cytokeratin 18 (CK18) and Vimentin. Some tumor cells were positive for CD117 (c-kit), whereas all of them were negative for CK7, CK20, Desmin and CD34  DD contributed in writing and revision of the manuscript. All authors read and approved the final manuscript.


P < 0.001), although the two compartments were also significantly correlated with each other. Tumours with low proliferation at the invasive margin had a significantly poorer prognosis both in univariate (P = 0.014) and in multivariate survival analyses (P = 0.042). We conclude that Dukes' B colorectal cancers exhibit a systematic spatial heterogeneity with respect to proliferation, and tumours with low proliferation at the invasive margin had a poor prognosis. The present data independently confirm recent results from the authors, and provide new insights into the understanding of tumour cell proliferation in colorectal cancer. © 1999 Cancer Research CampaignThe conflicting results about the prognostic impact of tumour cell proliferation in colorectal cancer might be explained by the heterogeneity observed within these tumours. We have investigated whether a systematic spatial heterogeneity exists between different compartments, and whether the presence of such a systematic heterogeneity has any impact on survival. Fifty-six Dukes' stage B colorectal cancers were carefully morphometrically quantified with respect to the immunohistochemical expression of the proliferative marker Ki-67 at both the luminal border and the invasive margin. The proliferative activity was significantly higher at the luminal border compared with the invasive margin (


Superior vena cava (SVC) and inferior vena cava (IVC) pressures were measured serially duringlaparoscopic cholecystectomy in which the intra-abdominal pressure was maintained at12mmHg. The influences of alteration of position from 15 degrees head-down to 15 degreeshead-up and of the operative procedure of holding the gallbladder up to the right subphrenicspace on SVC and IVC pressures were mild. IVC pressure was maintained almost equal to theintra-abdominal pressure during prolonged continuous pneumoperitoneum lasting longer than60min, while SVC pressure did not change significantly during operation. The discrepancybetween SVC and IVC pressures underwent no change during continuous pneumoperitoneum.


This study presents a retrospective review of chest radiography in children with Murphy stage III T-cell lymphoblastic lymphoma. All received a standard leukaemia-based protocol with intensive induction, consolidation and continuing chemotherapy. Neither initial thoracic disease bulk nor the presence of a pleural effusion predicted outcome. However a significant difference was found when the 50 patients in whom the chest radiograph returned to normal within 60 days of commencing treatment were compared with the 18 patients with persistent mediastinal abnormalities, for both event-free  and overall survival . It appears that this relatively simple estimate of chemosensitivity may identify a group of particularly good-risk patients in whom drugs associated with late morbidity such as anthracyclines may be reduced and conversely a higher risk group in whom further intensification of treatment would be justified.


Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable anti-tumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 10(4)-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia coli (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.


To the Editor: Multidrug-resistant Shigella dysenteriae type 1 caused an extensive epidemic of shigellosis in eastern India in 1984 , and the characteristic colonies were identified by standard biochemical methods  yielded S. dysenteriae type 1 occurred in the northern part of West Bengal in eastern India among tea garden laborers from April 2002 to May 2002; 1,728 persons were affected (attack rate of 25.6%). Sixteen persons died. The isolated S. dysenteriae type 1 strains were found intermediately sensitive to fluroquinolones with an MIC of 2 μg/mL .A similar outbreak of This drug-resistant Shiga bacillus is highly likely to spread further and will certainly pose a major therapeutic challenge unless adequate preventive measures are immediately instituted to contain its spread. Appropriate awareness programs for the community and reorientation training for physicians and other health personnel would be helpful to prevent further transmission of these resistant organisms. Alternative drugs to treat drug-resistant cases and an effective vaccine are also needed.


To our knowledge we report the first case of percutaneous mechanical thrombectomy used for the treatment of massive pulmonary embolism in the United Kingdom. Pulmonary embolism is a common disease process but can be difficult to diagnose. Massive pulmonary embolism presenting with profound hypotension, however, is rare. Both phenomena carry with them significant mortality. Traditionally those patients suffering haemodynamic compromise from pulmonary embolism are treated with intravenous or catheter-directed thrombolysis. When this is contraindicated surgical embolectomy or mechanical techniques via a right heart catheter are alternative options. The former is well established but the latter is less commonly utilised in clinical practice. Our aim is to highlight the effectiveness and relative safety of percutaneous mechanical thrombectomy as a therapeutic tool in massive pulmonary embolism.A 70 year-old gentleman presented with a 4-month history of dry cough and general malaise. Clinical examination along with routine chest radiograph confirmed a left pleural effusion which was drained. Computed tomography of the chest, abdomen and pelvis revealed a left renal mass consistent with renal cell carcinoma plus multiple metastatic subpleural nodules. Following planned thoracoscopy and pleural biopsy the patient became acutely dyspnoeic and hypotensive. Relevant investigations including computed tomography pulmonary angiogram confirmed a large saddle embolus extending in to the lobar branches of both left and right pulmonary arteries. There were several relative contraindications to thrombolysis and so the patient proceeded to have percutaneous mechanical thrombectomy with excellent results. The patient made a full recovery from the acute episode and was discharged home on warfarin with a view to planned cyto-reductive nephrectomy.We illustrate here that percutaneous mechanical thrombectomy can be a safe and effective method of treating massive pulmonary embolism when thrombolysis is relatively contraindicated. It may also be of use as an adjuvant therapy in those patients able to receive thrombolysis. In the future further evaluation involving a larger cohort of subjects is necessary to determine whether this treatment is superior to surgical embolectomy when thrombolysis cannot be performed. Pulmonary embolism (PE) is a common but under-diagnosed disease process with an estimated incidence of about 60–70 per 100 000 of the general population. Here theDespite a lack of robust data British Thoracic Society (BTS) guidelines published in 2003 clearly recommend the use of thrombolysis in massive PE; the earlier the better.[A 70 year-old male presented with a 4-month history of dry cough and general malaise. He had had a right arm melanoma excised 5 months previously but otherwise had no significant medical history. Clinical examination revealed a left pleural effusion confirmed by chest radiograph which subsequently drained blood-stained fluid positive for reactive mesothelial cells and lymphocytes. Cytology was negative for malignant cells. Computed tomography (CT) of his thorax and abdomen undertaken just prior to discharge from hospital revealed a left renal mass consistent with renal cell carcinoma (RCC) and multiple bilateral subpleural nodules. Renal function was normal at this stage.The patient was electively re-admitted a week later for planned thoracoscopy and pleural biopsy. Biopsy confirmed the subpleural nodules to be metastatic RCC deposits. Following repeat chest drain insertion the patient collapsed some hours later and became acutely dyspnoeic with oxygen saturations of 82% on room air and a systolic blood pressure of 96 mmHg. There was no clinical evidence of lower limb deep vein thrombosis. Arterial gases confirmed type I respiratory failure and an electrocardiogram revealed a sinus tachycardia with new right bundle branch block. Subsequent CT pulmonary angiogram (CTPA) demonstrated a large saddle embolus with thrombus extending into the lobar branches of both main pulmonary arteries Fig. . MassiveWe proceeded to rheolytic thrombectomy using the AngioJet system . Access was gained via 5-French (F) and 10F sheaths into the right femoral vein. A 4F sheath was also introduced into the right femoral artery for blood pressure monitoring. We confirmed the absence of thrombus in the inferior vena cava (IVC) angiographically before proceeding with right heart catheterisation. A 0.035-inch guidewire was placed in the right ventricle over which a 5F multipurpose-1 (MP-1) diagnostic catheter was fed. Pulmonary angiography via this catheter revealed filling defects in both pulmonary arteries  declare that they have no competing interests.AM assisted with the actual procedure, helped to acquire data and images for figure presentations, drafted the manuscript and revised it critically for important intellectual content. AA conducted the actual procedure and gave a critical appraisal of the draft manuscript. All authors read and approved the final manuscript.Selective pulmonary angiogram pre-thrombectomy. Selective pulmonary angiogram demonstrating evidence of thrombus material in the left main pulmonary artery prior to percutaneous mechanical thrombectomy.Click here for fileSelective pulmonary angiogram post-thrombectomy. Selective pulmonary angiogram demonstrating restoration of blood flow in the left main pulmonary arterial tree following percutaneous mechanical thrombectomy.Click here for file


When normal rat and mouse embryo cells were treated with a cell free extract of S37 ascites tumour, morphological transformations occurred in both. The transformed cells readily induced lymphosarcoma-type tumours in the mice inoculated when newborn or young adults (8-12 weeks old), but not so readily in rats.Various tests carried out with these cells strongly indicate the presence of an oncogenic and transmissible agent in S37 ascites tumour. This agent appears to be related to mouse sarcoma virus isolated from animals with Moloney leukaemia but differs in only producing characteristic lymphosarcoma-type tumours.


Guinea pig and mouse peritoneal macrophages formed antibody-dependent rosettes with guinea pig L2C leukaemic cells, but were unable either to phagocytose the cells or to kill them extracellularly as judged by the retention of 51Cr. Macrophages previously activated by BCG in vivo also failed to exhibit phagocytosis or cytoxicity towards the antibody-coated cells. These failures could not be attributed to deficient function of the macrophages nor to antigenic modulation of the L2C cells. The antibodies involved were capable of mediating lysis by complement, and ADCC by human leukocytes. However macrophages were cytostatic to antibody-coated L2C cells in that uptake of 3H-thymidine or 3H-deoxycytidine was abruptly and in some cases completely inhibited upon cell contact being established. Antigenic modulation which had proceeded sufficiently to protect against lysis by complement did not protect against cytostasis. Syngeneic macrophages had greater cytostatic activity than did allogeneic or xenogeneic. Macrophage activation by BCG did not result in significantly increased cytostasis. A univalent antibody derivative Fab/c was also capable of mediating cytostatis by the macrophages.


Metastatic malignancy to the thyroid gland is generally uncommon due to an unfavourable local thyroid micro-environment which impairs the ability of metastatic cells to settle and thrive. Metastases to the thyroid gland have however been reported to occur occasionally particularly if there has been disruption to normal thyroid tissue architecture.18F-FDG PET scan positive thyroid nodule which was subsequently confirmed at surgery to be a focus of metastatic rectal adenocarcinoma within a primary poorly differentiated papillary thyroid carcinoma.We report a patient with a history of surgically resected rectal adenocarcinoma who presents with a rising serum CEA level and an Subsequent treatment involved right hemi-thyroidectomy, pulmonary wedge resection of oligometastatic metastatic colorectal cancer and chemotherapy.Metastatic rectal carcinoma to the thyroid gland and in particular to a primary thyroid malignancy is rare and unusual. Prognosis is likely to be more dependent on underlying metastatic disease rather than the primary thyroid malignancy hence primary treatments should be tailored towards treating and controlling metastatic disease and less emphasis placed on the primary thyroid malignancy. Metastatic malignancy to the thyroid gland, although considered rare, occurs more frequently than expected. Microscopic metastases to the thyroid gland have been reported to occur in 4%–9% of autopsy studies,2, with A 52 year old man with a history of T3N1M0 (Dukes C) rectal adenocarcinoma treated with neo-adjuvant chemo-radiotherapy (5FU + radiotherapy) followed by anterior resection and adjuvant chemotherapy (5FU) presented 18 months post completion of therapy with rising serum CEA level  and a 15 mm left lower lobe pulmonary nodule on computerized tomography (CT), suggestive of a metastatic deposit (figure 18F-FDG whole body Positron Emission Tomography (PET) scan was performed to further evaluate the nature of the left lower lobe pulmonary nodule and to evaluate any other possible sites of metastatic disease. The whole body PET scan demonstrated significant 18F-FDG uptake in the left lower lobe pulmonary nodule compatible with a metastatic deposit (figure An t figure . An inteAn ultrasound guided fine needle aspirate of the right lower pole thyroid nodule was performed which revealed malignant cells with features suggestive of a primary papillary thyroid cancer. A right hemi-thyroidectomy was subsequently performed. Macroscopically, the resected thyroid specimen demonstrated a well-circumscribed dominant thyroid nodule measuring 32 mm in diameter with a pale tan capsule less than 1 mm in thickness. The cut surface had a variegated appearance with pale tan friable tissue intermixed with foci of yellow tissue and dark brown foci. Histological examination revealed cells typical of metastatic adenocarcinoma of the colon intermixed in a background of a poorly differentiated papillary thyroid carcinoma (figure A wedge resection of the left lower lobe pulmonary nodule was subsequently performed which confirmed metastatic colorectal adenocarcinoma figure . Serum CMetastatic lesions to the thyroid gland are generally considered rare, possibly due to a high oxygen and iodine environment which may impair the ability of metastatic cells to settle and develop. Abundant high velocity blood flow through the thyroid gland also possibly plays a role in impeding the ability for metastatic cells to gain a foothold. Perhaps18F-FDG PET, an increasing number of incidental cases of metastatic disease to the thyroid gland are likely to be detected. Well differentiated primary thyroid carcinomas such as papillary and follicular carcinomas are generally not 18F-FDG avid on PET scanning and usually only become 18F-FDG avid if they de-differentiate.With the advent of improved diagnostic imaging technology such as 18F-FDG avid primary thyroid malignancies are generally a more aggressive variant of primary thyroid cancer which harbour a higher rate of unfavourable prognostic factors and are often less well differentiated[18F-FDG avid.It has been reported in the literature incidentally PET detected To our knowledge there have been no cases of metastatic malignancy within a primary thyroid malignancy reported previously in the literature. As such, there is scant evidence in the literature regarding the most appropriate management strategy for such a patient. Conventional management of a primary thyroid malignancy usually involves total surgical thyroidectomy followed by radioiodine therapy to ablate the thyroid remnant, decrease risk of recurrence and enable adequate follow up using I-131 whole body scintigraphy and stimulated thyroglobulin levels -13.In our case, prognosis is more likely to be dependent on the patient's metastatic rectal adenocarcinoma rather than the primary thyroid malignancy. Stage IV metastatic rectal adenocarcinoma portends a poor prognosis, with five year survival rates of between 4%–8%,15. MoreAs a result, the primary treatment focus in our patient was tailored towards treating metastatic rectal carcinoma and less so the primary thyroid malignancy. It is also of note, many poorly differentiated thyroid carcinomas are not particularly radioiodine avid and it is quite possible the impact of radioiodine therapy on reducing recurrence rates may be greatly diminished in this setting.Metastatic rectal carcinoma to the thyroid gland and in particular to a primary thyroid malignancy is uncommon and to our knowledge has not been reported. Prognosis is likely to be more dependent on underlying metastatic disease rather than the primary thyroid malignancy hence primary treatments should be tailored towards treating and controlling metastatic disease and less emphasis placed on the primary thyroid malignancy.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.MC conceived the idea, reported the PET scan, performed the literature search, provided the radiographic images and drafted the manuscript. MM administered chemotherapy, reviewed and revised manuscript, JS performed surgery, reviewed and revised manuscript, SS reported histopathology, provided histopathological images, reviewed and revised manuscript and DT treated patient, reviewed and revised manuscript. All authors have read and approved the final manuscript.


Generalized lymphosarcomatosis (leukaemia) of non-thymic type occurs in mice bearing 90Sr or 239Pu or 226Ra. Tumours passaged from such mice have been tested for tumour-associated transplantation antigens that could provoke a protective immunity which would be expected if such antigens were determined by virus activated by the irradiation. Sub-threshold doses of living syngeneic tumour, large doses of living allogeneic tumour and large doses of killed syngeneic tumour were without protective effect. This suggests that viruses observed electron micrographically in such tumours are passengers and not causative.


The relationship of testicular seminoma with several factors was explored using a case-control study. Previously recognized associations with cryptorchidism and infantile inguinal hernia were confirmed and relationships were also found with cycling and horse-riding. These findings represent the first relationships of testicular cancer with well-defined postnatal risk factors.


Heat shock proteins have been reported to confer resistance to certain antineoplastic drugs. We investigated the impact of hsp70 overexpression on the efficacy of two new anti-cancer drugs, topotecan and gemcitabine. We used the fibrosarcoma WEHI-S cells stably transfected to overexpress the hsp70 cDNA from the constitutive SV40 promoter and appropriate control cells. After topotecan and gemcitabine treatment hsp70-overexpressing cells showed a marked elevation in cell survival, suggesting that hsp70 overexpression was sufficient to confer resistance to the drugs. In addition, hsp70-overexpressing cells were capable of starting cell proliferation after treatment with drug dosages that were lethal to control cells. Our results demonstrate that hsp70 overexpression represents a possible cause of drug resistance. In order to transfer these data to tumour cells constitutively expressing stress hsp70 due to the constitutive activity of the original hsp70 promoter we sought to supress the heat shock response pathway by the natural flavonoid quercetin, known to inactivate the heat shock transcription factor (HSF). Using a suitable cell line, we demonstrated the sensitising activity of quercetin. We found that antineoplastic drug concentrations exerting cytotoxic activity were markedly lower when cells were pretreated with quercetin. Concomitantly, hsp70 expression was strongly down-regulated under quercetin treatment. Our data indicate that quercetin may be useful as a sensitiser in chemotherapeutically treated patients suffering from hsp70-overexpressing tumours.


Evidence increasingly shows that stream ecosystems greatly contribute to global carbon fluxes. This involves a tight coupling between biofilms, the dominant form of microbial life in streams, and dissolved organic carbon (DOC), a very significant pool of organic carbon on Earth. Yet, the interactions between microbial biodiversity and the molecular diversity of resource use are poorly understood.Using six 40-m-long streamside flumes, we created a gradient of streambed landscapes with increasing spatial flow heterogeneity to assess how physical heterogeneity, inherent to streams, affects biofilm diversity and DOC use. We determined bacterial biodiversity in all six landscapes using 16S-rRNA fingerprinting and measured carbon uptake from glucose and DOC experimentally injected to all six flumes. The diversity of DOC molecules removed from the water was determined from ultrahigh-resolution Fourier Transform Ion Cyclotron Resonance mass spectrometry (FTICR-MS). Bacterial beta diversity, glucose and DOC uptake, and the molecular diversity of DOC use all increased with increasing flow heterogeneity. Causal modeling and path analyses of the experimental data revealed that the uptake of glucose was largely driven by physical processes related to flow heterogeneity, whereas biodiversity effects, such as complementarity, most likely contributed to the enhanced uptake of putatively recalcitrant DOC compounds in the streambeds with higher flow heterogeneity.Our results suggest biophysical mechanisms, including hydrodynamics and microbial complementarity effects, through which physical heterogeneity induces changes of resource use and carbon fluxes in streams. These findings highlight the importance of fine-scale streambed heterogeneity for microbial biodiversity and ecosystem functioning in streams, where homogenization and loss of habitats increasingly reduce biodiversity. A growing body of reviews and meta-analyses has recently highlighted the functional consequences of biodiversity for ecosystem functioning In this paper, we report the results of an experiment designed to test the hypothesis that fine-scale physical heterogeneity in streamside flumes affects bacterial biodiversity and DOC resource use by benthic biofilms. Biofilms are matrix-enclosed and attached communities that dominate microbial life in streams where they can control key ecosystem functions −1). The resulting flow may be identified as shallow rough-bed flow typical for headwater streams. The flumes were continuously fed in a once-through mode to assure identical water chemistry and microbial inoculum. A header tank and tilting weirs ensured steady and spatially uniform flow. Nearly identical average flume-level velocities were achieved by adjusting flume slopes individually with repeated measurements of mean velocity by slug additions of NaCl-solutions Experiments were conducted in streamside flumes . Five flumes contained 38 triangular dune-shaped bedforms , respectSDRxyz, the standard deviation of Rxyz is the central independent variable and a surrogate for flow heterogeneity.Acoustic Doppler Velocimetry  described the 3-D flow fields and served to derive heterogeneity of flow as its spatial variation. In each flume, velocity was mapped over one entire bedform (∼5 mm above substrate) at nodes of a 5×5 cm grid. For each node, we determined depth, mean length of the 3-dimensional velocity vector y TI see . 80 measa) and for bacterial community composition. Coupons were sampled four times over a growth period of 4–5 weeks during summer season. Biofilms reached stationary growth after approximately 3–4 weeks. Coupons from triplicate bedforms were processed as composite samples for bacterial community composition. To determine microbial biomass SYTOX-stained bacteria were counted and sized using a Cell-Lab-Quanta (Beckman Coulter). Chlorophyll-a was assayed as described elsewhere Sterile unglazed ceramic coupons (1×2 cm) served as substratum for biofilm growth DNA from biofilm communities was extracted and purified with the UltraClean Soil DNA Isolation kit . Non-colonized ceramic coupons served as negative controls. The fluorescently labeled primers used for PCR of the 16S rRNA gene were FAM labeled 27F and JOE-labeled 1492R  q = 1D)/ln(S)ln to study dynamics of complex DOC. This DOC from riparian vegetation mimics an important terrestrial subsidy, which can generate substantial pulses of DOC in headwaters −1 and 8 mg L−1 for glucose and DOC, respectively. Water samples were collected at the flume outlets, filtered and frozen immediately before analysis. Glucose was analysed with HPLC–PAD (Dionex ICS3000) using a CarboPac PA20 column . DOC concentration was measured using a SIEVERS (Ionics Instruments) and a Shimadzu TOC-VCPH/CPN. Mass transfer coefficients  for glucose and bulk DOC were calculated from log-linear fits of concentration decline in time  from riparian vegetation (time see .100O80N5S. See SDRxyz). See DOC (2 L water samples) from the header tank (inflow) and from each flume after recirculation  was extracted using XAD-8 resin 5 permutations. All calculations were done in R We hypothesized biodiversity components and community composition as explanatory links between environmental heterogeneity and resource use. Because of the multivariate nature of most of the involved entities, we used causal modeling on dissimilarity matrices calculated from subsets of appropriate variables −1 to 6.3 cm s−1 across all six flumes (−1 which translates to a residence time of 8.8 min) and mean water depth (6.7±0.4 cm) among flumes  whereas bacterial abundance did not . Thus, we could identify a relatively small but stable beta-component that is clearly related to the hydrodynamic microenvironment and largely relies on relative abundance patterns of frequent OTUs.T-RFLP analyses identified a total of 76 bacterial operational taxonomic units (OTUs) in the biofilm samples in all flumes with an average of 26±13 (mean±SD) per flume. Total bacterial diversity at flume-scale  was partitioned into the two orthogonal independent components alpha (average diversity within microhabitats) and beta (among microhabitats) diversity, and into richness and evenness, respectively see . We emplTo explore the possible consequences of spatial flow heterogeneity and related biodiversity patterns for resource use as an ecosystem process, we first measured the uptake of experimentally added monomeric glucose and then of a complex DOC mixture by mature biofilms typical for baseflow in headwaters. We extracted DOC from riparian vegetation to simulate a major carbon input to streams during leaf litter fall in autumn vf, see vf is independent from scaling effects of velocity and flume depth; its product with concentration represents the average flux of a solute to the streambed. We found vf of glucose (vf-Glucose) and bulk DOC (vf-DOC) significantly increased with increasing flow heterogeneity  using ultrahigh-resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry  exhibited less variation at higher flow heterogeneity, indicating more even  consumption across all compounds of the DOC mixture (To test whether the observed increase in  mixture . This wo mixture  indicaterIinflow) as a key predictor of compound-specific mass transfer coefficients (vf-indDOC) in individual flumes; followed by the O∶C ratio, H∶C ratio and aromaticity (rIinflow) are preferentially removed from the stream water – decoupled from their absolute uptake proportional to concentration . These results also suggest that more oxygenated compounds are removed more readily from the water. Specifically, regression coefficients of the O∶C ratio, H∶C ratio and aromaticity  decreased with increasing flow heterogeneity. Similarly, negative relationships between slopes of predictors (or predicted effect sizes) of individual flume models and flow heterogeneity were found for O∶C, aromaticity and nitrogen  with higher heterogeneity  or intrinsic chemical properties — at higher flow heterogeneity. Hence, we further used the model coefficients as a proxy for resource use diversity.In support of the evenness pattern , the strnitrogen . In the The typical way of qualitatively visualizing ultrahigh-resolution spectra are Van Krevelen diagrams showing compounds in a chemical space defined by H∶C versus O∶C vf-indDOC and rIinflow (as a proxy for resource use diversity), which correlates better with bacterial beta diversity than with flow heterogeneity  constant . Similarly, the standard deviation of vf-indDOC, an additional descriptor for resource use diversity, correlated with beta diversity . Neither gamma diversity nor gamma evenness explained the diversity in resource use.Our experiments revealed a significant positive relationship between bacterial biodiversity and the diversity of DOC compounds removed from the water. This is supported by the slope of the relationship between To formally test biodiversity and community composition as explanatory links between flow heterogeneity and resource use, we applied causal modeling on dissimilarity matrices  and compIn this study, we present evidence that the manipulation of fine-scale flow heterogeneity may affect bacterial biodiversity in benthic biofilms and their resource use. The more heterogeneous flow landscapes triggered larger bacterial biodiversity and structural differentiation of biofilms, ultimately resulting in a broader range of DOC compounds removed more rapidly by the biofilms. To our knowledge, this is the first study attempting to integrate microbial diversity with environmental heterogeneity and chemical diversity of DOC as the major resource for microbial heterotrophs.Ecological theory Our results from causal modelling indicate that complementarity as a mechanism underlying the relationship between biodiversity and resource use may be true for DOC as a complex resource but most likely not for glucose. Rather, we argue that glucose uptake is largely controlled by variables modulated by flow heterogeneity, such as turbulence In contrast, we postulate that DOC removal was most likely controlled by mechanisms related to biodiversity rather than just to flow. Several lines of evidence support this hypothesis. The increase of bacterial beta diversity across the gradient of flow heterogeneity suggests diversification of hydrodynamic microhabitats along bedforms to generate distinct local communities assembled from the same species pool in the streamwater vf-indDOC of 361 compounds) exhibiting less variation at higher flow heterogeneity, which indicates more even  consumption across all compounds of the DOC pool. This would translate into decreasing metabolic specialization as flow heterogeneity increases. Evenness patterns of the molecular composition of both the inflow and residual DOC pools  enabling them to maximize resource use and to maintain high biomass irrespective of the hydrodynamic conditions Patterns of bacterial abundance across the six heterogeneity landscapes did not parallel patterns of glucose and DOC removal, indicating no role of bacterial abundance as a driver of resource use. We did not quantify the architectural differentiation of biofilms across flumes, but it becomes obvious from the spatial variation of both chlorophyll-The observed gradients of mass transfer and resource use diversity were not generated by surface area effects in the flumes. In fact, mass transfer coefficients are expressions of uptake performance corrected for the water volume and streambed surface area in the various flumes. Furthermore, bedforms were sealed to avoid confounding effects from varying streambed topography and induced hydrodynamic exchange and carbon processing What are the implications of our study? Streams and rivers are increasingly recognized to contribute to global carbon fluxes, but the underlying mechanisms remain obscure Figure S1Descriptive statistics of depth and velocity across the flow heterogeneity gradient.(0.02 MB PDF)Click here for additional data file.Figure S2Van Krevelen diagrams of CHONS molecular compositions.(0.14 MB PDF)Click here for additional data file.Figure S3Relationships of beta-diversity with diversity of DOC resource use and bulk DOC uptake.(0.02 MB PDF)Click here for additional data file.Figure S4Alternative metavariable path diagrams calculated from Mantel statistics.(0.03 MB PDF)Click here for additional data file.Table S1Characterization of the hydrodynamic conditions over the control streambed and the bedforms of the landscapes increasing in flow heterogeneity.(0.04 MB PDF)Click here for additional data file.Table S2Water-related environmental background conditions  measured at the outlet of mesocosms and in the header tank.(0.01 MB PDF)Click here for additional data file.Table S3Metavariable dissimilarity (distance) matrices and underlying variables.(0.02 MB PDF)Click here for additional data file.Table S4Results of hierarchical partitioning analysis of DOC compound-specific characteristics as predictors for mass transfer to the streambed.(0.01 MB PDF)Click here for additional data file.Table S5Selected multiple linear regression models using DOC compound-specific characteristics as predictors for mass transfer to the streambed.(0.01 MB PDF)Click here for additional data file.Table S6Results of flume level (gamma) biodiversity partitioning into independent orthogonal components alpha and beta, or richness and evenness.(0.02 MB PDF)Click here for additional data file.Table S7Associations between metavariable dissimilarity matrices as expressed by Mantel statistics.(0.02 MB PDF)Click here for additional data file.Text S1Method Details(0.11 MB PDF)Click here for additional data file.


Reactivity of the monoclonal antibody 791T/36 with secondary malignant deposits has been investigated in frozen sections of 74 human liver biopsy specimens. There was no reactivity with hepatocytes but in some instances binding to fibrous tissues and in one case to portal tract lymphocytes was observed. Sections from 9 biopsy specimens contained malignant deposits. In seven of these 791T/36 bound either to malignant cells or to pseudoacinar contents . Two undifferentiated tumours  showed no antibody binding. The histological pattern of reactivity previously reported with primary tumours appears to be similar in secondary deposits. A wider range of tumours than recognised hitherto binds 791T/36. Whether the binding to fibrous tissue seen in some instances is sufficient to cause diagnostic uncertainty when 791T/36 is used for scanning requires further investigation.


Reverse transfection microarrays were described recently as a high throughputmethod for studying gene function. We have investigated the use of this technology fordetermining the subcellular localization of proteins. Genes encoding 16 proteins witha variety of functions were placed in Gateway expression constructs with 3′ or 5′ greenfluorescent protein (GFP) tags. These were then packaged in transfection reagent andspotted robotically onto a glass slide to form a reverse transfection array. HEK293Tcells were grown over the surface of the array until confluent and GFP fluorescencevisualized by confocal microscopy. All C-terminal fusion proteins localized to cellularcompartments in accordance with previous studies and/or bioinformatic predictions.However, less than half of the N-terminal fusion proteins localized correctly. Of thosethat were not in concordance with the C-terminal tagged proteins, half did not exhibitexpression and the remainder had differing subcellular localizations to the C-terminalfusion protein. This data indicates that N-terminal tagging with GFP adversely affectsthe protein localization in reverse transfection assays, whereas tagging with GFP atthe C-terminal is generally better in preserving the localization of the native protein.We discuss these results in the context of developing high-throughput subcellularlocalization assays based on the reverse transfection array technology.


Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions.Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt.We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology. In this paper we present a molecular diagnostics method that combines isothermal amplification of target tmRNA molecule with microarray-based detection. Nucleic Acid Sequence Based Amplification (NASBA) is a sensitive isothermal RNA amplification technology  that offgnostics ,14. AminS. pneumoniae tmRNA molecules was verified by observing only one peak of predicted size (307 nucleotides) nucleic acid on RNA 6000 chip electropherogram. Respective microarray signal intensities of labeled NASBA products are shown on Fig. Addition of aaUTP to NASBA mix resulted in a concentration-dependent effect on the reaction performance. Concentrations of up to 0,5 mM for sodium salt and 1 mM for lithium salt did not influence NASBA efficiency as seen from the amount of RNA produced Fig. , whereasThe aim of this work was to improve and expand the potential of a combined NASBA-microarray approach by developing a new optimized protocol suitable for further possible use in microbial diagnostics. All together, we have introduced a new method suitable for high-speed parallel detection of RNA targets. NASBA technology, widely used in various microbiology laboratories all over the world, is for the first time combined with microarray based RNA detection technology using simple one-step NASBA product labeling method. Previous methods described so far for NASBA amplicon detection have used additional enzymatic steps after the amplification and secondary labeled probes ,5. In ouIn general we have presented here a new optimized protocol for highly potential microarray based detection of NASBA amplified RNA products. Our method is suitable for further possible diagnostic applications where rapid identification of various RNA molecules from bacteria or different microorganisms is needed. The combination of NASBA with microarray detection is particularly advantageous in settings where different bacterial species may be present  or in clinical settings where it is necessary to identify one particular infection causing species from a panel of potential pathogens.Streptococcus pneumoniae strain ATCC 33400  was grown in Brain Heart Infusion Broth . Total RNA was purified from overnight cultures using RiboPure Yeast Kit . Although the RiboPure Yeast kit is not originally intended for RNA extraction from bacteria, high quality bacterial total RNA was obtained without making any modifications to manufacturer's instructions.S. pneumoniae total RNA. Conventional NASBA, in which the T7 promoter is included in the reverse primer, generates an antisense RNA product [NASBA primer pair Table  was desi product . In orde product . NASBA r2CO3 and incubated for one hour with 3 nmoles of amino reactive Cy3-N-hydroxysuccinimide ester dye  dissolved in 0,5 μl DMSO . Excess dye was quenched by addition of 3,5 μl 4 M hydroxylamine solution . Labeled RNA was purified with NucleoSpin kit and quantified using Bioanalyzer 2100 platform with RNA 6000 Nano Chip  according to the manufacturers manual.Following amplification, NASBA products were purified with NucleoSpin RNA Clean-Up kit  and vacuum dried. Labeling of the RNA product was carried out using protocol published by 't Hoen and colleagues with some modifications . The RNAS. pneumoniae tmRNA sequence . Probes were ordered from Metabion  and spotted onto SAL-1 Ultra microarray slides by Asper Biotech .The custom made microarray contained 97 probes, covering whole S. pneumoniae specific microarray in an automated HS-400 hybridization station  at 34°C. Complete hybridization protocol is shown in Table One third of the labeled NASBA product was resuspended in 80 μl of microarray hybridization buffer and hybridized for four hours on the Slides were scanned using Affymetrix 428 scanner  λ = 532 nm. Raw signal intensity data was analyzed with Genorama software (Asper Biotech). Fixed concentration of short fluorescent oligonucleotide control sequences ('spikes'), complementary to the control oligonucleotides on microarray, were added to the final 80 μl hybridization solution for normalization purposes. At first step all signals from all microarrays, obtained from dilution series experiment, were rescaled by equating the average of spike-specific signals. Secondly, to compare data from 97 different probes on single microarray, all individual signal intensities were divided with the corresponding probe specific average signal intensity calculated over all microarrays in one replicate experiment. Average over probe-wise normalized signal intensities  were then calculated for each dilution series point (for each single microarray). Finally, considering replicate experiments (at least three), confidence intervals (± one SD) were calculated for each average dilution series point (for every different aaUTP concentration).OS carried out the NASBA and microarray experiments, conducted statistical analysis and drafted the manuscript. BG carried out the microbiological experiments and RNA extraction and helped to draft the manuscript. SP participated in NASBA and microarray experiments and helped to draft the manuscript. KT participated in NASBA and microarray experiments. PP designed the microarray and helped to draft and review the manuscript. LK helped designing microarray and helped with data analysis. MR participated in design and coordination of making a microarray and helped conduct data analysis. MM conceived of the study and participated in its design and coordinated microbiological experiments. AK conceived of the study, participated in its design, coordinated NASBA and microarray experiments and helped to draft the manuscript. All authors read and approved the final manuscript.Microarray probes used in experiment. Table showing all the 97 microarray probes (covering whole S. pneumoniae tmRNA sequence) and their characteristics.Click here for file


Authors describe the incremental value of hybrid SPECT-CT in upstaging disease and changing the management strategy of a case of renal cell carcinoma Bone scintigraphy is less sensitive for detection of osteolytic lesions. We describe the utility of hybrid modality SPECT-CT in demonstrating the osteolytic lesion on morphological (CT) component of SPECT-CT associated with focal subtle tracer uptake.‐3We report the case of a 47-year-old male patient who presented with abdominal pain, lower urinary symptoms and hematuria for three months. He was diagnosed as a case of renal cell carcinoma and subjected to radical nephrectomy. The patient also underwent a whole body bone scan for a metastatic workup. A planar whole body bone scan along with a hybrid single photon emission computed tomography (SPECT) / Computed Tomography (CT) imaging was performed. The planar imaging showed an area of mildly increased tracer uptake in the second lumbar vertebra . Subsequ


Plasmodium falciparum mitochondria and is the focus of a drug discovery programme. A miniaturised assay for recombinant PfNDH2 with robust assay performance measures was generated for the high throughput screening (HTS) of a focused library of 17,000 drug-like compounds. A quantitative structure-activity relationship has been developed around one of the chemical templates derived from the HTS hits. Lead molecules developed to date show selective inhibitory activity against PfNDH2 versus P. falciparum bc1 or dihydroorotate dehydrogenase (DHODH). Potent enzyme inhibition is accompanied by in vitro parasite kill of multidrug-resistant strains in the low nM range and clearance of parasites from in vivo P. berghei models. Lead molecules also display excellent in vitro therapeutic indices against human cell lines and bovine bc1. Initial metabolic studies in human liver microsomes and hepatocytes indicate favourable pharmacology. These data support the further development of this new candidate drug targeting a novel parasite component.NADH:quinone oxidoreductase (PfNDH2) represents a metabolic choke point in the respiratory chain of


Tumour growth depends on neovascularisation and tumour cell proliferation. Factor VIII-related antigen (F-VIII RA) localises to vascular endothelium. Expression of proliferating cell nuclear antigen (PCNA) is correlated with cell proliferation. We investigated the correlation between the expression of these antigens and prognosis in gastric carcinoma. A total of 108 specimens resected from patients with gastric carcinoma were investigated by staining with monoclonal antibodies against F-VIII RA and PCNA. Microvessel count  and PCNA labelling index  were determined. The results showed that prognosis was significantly worse in patients who had a tumour with a high MVC (16 or greater) or a high PCNA LI (42% or greater) than in those patients who had a tumour with a low MVC (less than 16) or a low PCNA LI (less than 42%). Furthermore, MVC was significantly associated with the risk of hepatic recurrence. In conclusion, both MVC and PCNA LI may be good prognostic indicators in patients with gastric carcinoma.