• A study of childhood renal tumours using xenogeneic antiserum.

      Kumar, Shant; Marsden, Henry B; Kumar, P (1980-08)
      Fifty-six primary childhood renal tumours, 14 normal and 12 fetal kidneys were examined for their staining reaction with xenogeneic anti-Wilms' antiserum. The antiserum was raised by injecting Wilms' tumour extracts into 6-month-old rabbits which had been rendered tolerant in utero with pooled normal kidney extracts. Renal carcinomas, mesoblastic nephroma and a large proportion of tubular wilms' tumours were stained by the antiserum. In contrast, 6 of 7 bone-metastasising renal tumours of childhood (BMRTC) failed to fluoresce when treated with the antiserum, suggesting that the BMRTC has a different origin from the other childhood renal tumours studied.
    • A study of cytokinetic amd motile prostate cancer cells using synchroton-based FTIR micro spectroscopic imaging

      Gazi, Ehsan; Dwyer, John; Lockyer, Nicholas P; Miyan, J; Gardner, Peter; Hart, Claire A; Brown, Michael D; Clarke, Noel W; School of Chemical Engineering and Analytical SCience, Faraday Building, The University of Manchester, PO Box 88, Manchester M60 1QD, UK (2005)
    • The study of haemopoietic stem cells in patients: concepts, approaches and cautionary tales.

      Testa, Nydia G; De Wynter, Erika A; Weaver, A; CRC Dept. of Experimental Haematology, Paterson Institute for Cancer Research, Manchester, U.K. (1996)
    • Study of innate immune responses during small cell lung cancer (SCLC) development

      Chudziak, Jakub; Galvin, Melanie; Banyard, Antonia; Simpson, Kathryn L; Frese, Kristopher K; Kilgour, Elaine; Hussell, T.; Dive, Caroline; Cancer Research UK Manchester Institute, Alderley Park, (2021)
      SCLC is a highly aggressive neuroendocrine tumor with dismal prognosis. Inability to detect SCLC early renders surgical resections a rare event, limiting investigation of the early stages of tumor development. We sought to address this challenge using a genetically engineered mouse model (GEMM) to investigate how the innate immune system responds to SCLC development. The RPM (Rb1fl/fl, p53fl/fl, MycLSL/LSL) SCLC GEMM was selected to characterize the local immune microenvironment throughout the process of tumor development. Following tumor induction using an intranasally installed Cre bearing adenovirus, lung tissue was collected and analyzed using multi-parametric flow cytometry to assess changes in innate immune cell populations. Induction of SCLC resulted in late stage disease being present in animals approximately 7 weeks after virus exposure. Flow cytometric analysis of immune cell populations of the whole lung showed no significant differences in the overall levels of immune cell types, although an upwards trend in the levels of neutrophils in tumor-bearing mice was observed. The most significant finding was a decrease in the expression levels of major histocompatibility complex class II (MHCII) in both alveolar and interstitial macrophage populations. Further characterization, including separate analysis of the two interstitial macrophage subpopulations described by Chakarov et al. (Science 2019), and Schyns et al. (Nature Communications 2019), showed significant drops in levels of MHCII in all populations as measured by median fluorescent intensity (MFI) (p=0.004 in alveolar macrophages, p=0.0148 in vascular supporting CD206+ve interstitial macrophages, and p=0.0027 in CD206-ve interstitial macrophages). The striking difference between the changes seen in these populations was that alveolar and CD206+ve interstitial macrophages both showed a downward shift in MHCII expression as a whole, while CD206-ve macrophages acquired a novel population completely negative for MHCII accounting for approx. half of the CD206-ve macrophage subpopulation. The development of SCLC has been found to result in all macrophage subtypes expressing lower levels of MHCII, pointing towards a more immunosuppressive immune environment. The population of CD206-ve MHCII-ve interstitial macrophages is of particular interest, as it is contrary to the currently described interstitial macrophage phenotypes. A more in-depth characterization of all macrophage populations in the mouse lung, including their precise phenotype and localization is currently underway using CyTOF and IHC approaches. The precise timings of the observed changes are being elucidated through analysis of a disease time-course ranging from very early stage to late stage disease, in order to better understand the process of SCLC development.
    • Study of interlaboratory reliability and reproducibility of estrogen and progesterone receptor assays in Europe. Documentation of poor reliability and identification of insufficient microwave antigen retrieval time as a major contributory element of unreliable assays.

      Rhodes, Anthony; Jasani, Bharat; Balaton, Andre J; Barnes, Diana M; Anderson, Elizabeth; Bobrow, Lynda G; Miller, Keith D; United Kingdom National External Quality Assessment Scheme for Immunocytochemistry and the Department of Histopathology, University College London Medical School, London, England. (2001-01)
      Immunohistochemical assays for estrogen receptors (ERs) and progesterone receptors (PRs) have not been surveyed for technical validity. In the present study, the reliability of the immunohistochemical assay for ER and PR was evaluated using data from 105 laboratories participating in external quality assessment (EQA) during a 2-year period. Technical variables associated with reliable immunostaining were analyzed. The efficiency of the antigen retrieval step was identified as the single most important contributory factor influencing the overall reproducibility of the assays. Reliable assays were found in 24 (36%) of 66 laboratories participating in continual EQA, including the majority of centers known to have clinically validated results. Inadequate assay sensitivity, with subsequent weak staining, was the main cause of poor and variable results by laboratories using microwave antigen retrieval; too short a heating time was identified as the principal contributory factor. Extension of the heating time resulted in significant improvement regardless of all other variables in the immunohistochemical protocol. Continual participation in EQA is an effective means for identifying and ameliorating variables that influence the reliability of immunohistochemical assays for predictive markers, thereby assisting in technical validation and standardization.
    • The study of multi-stage carcinogenesis in retinoblastoma and familial polyposis coli patient-derived skin fibroblast cell culture systems.

      Kinsella, Anne R; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, U.K. (1988-06)
      Carcinogenesis is considered to be a multi-step process comprising 'initiation', 'promotion' and 'conversion' events. Skin fibroblasts from patients with hereditary retinoblastoma (RB) and familial polyposis coli (FPC) were chosen for study since their predisposition to the tumour may be due to an inherited 'initiation' event which is present in every cell. Experiments involving skin fibroblasts from FPC patients showed certain of these cells to grow in semi-solid medium following treatment with the complete carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone. When tested prior to the commencement of the experiments the FPC patient cell populations had shown no strong predisposition to malignant transformation as assessed by increased saturation densities, reduced serum requirements, altered migration patterns in collagen gels, anchorage-independent growth and tumourigenicity in nude mice. Following carcinogen or promoter treatment, apart from exhibiting low-level frequencies of anchorage-independent growth, the cells appeared no more transformed than they were before. Parallel cytogenetic studies showed TPA to increase both tetraploidy and the chromosome-aberration frequency during the course of these transformation studies. However, the FPC cell clones induced by TPA to grow in semi-solid medium were, at best, considered to be only partially transformed when their properties were compared with those of tumour-derived cell lines.
    • A study of ovarian cancer patients treated with dose-intensive chemotherapy supported with peripheral blood progenitor cells mobilised by filgrastim and cyclophosphamide.

      Weaver, Andrew; Wrigley, E; Watson, A; Chang, James; Collins, Conor D; Jenkins, B; Gill, C; Pettengell, Ruth; Dexter, T Michael; Testa, Nydia G; et al. (1996-12)
      We have shown that large numbers of haemopoietic progenitor cells are mobilised into the blood after filgrastim [granulocyte colony-stimulating factor (G-CSF)] alone and filgrastim following cyclophosphamide chemotherapy in previously untreated patients with ovarian cancer. These cells may be used to provide safe and effective haemopoietic rescue following dose-intensive chemotherapy. Using filgrastim alone (10 micrograms kg-1), the apheresis harvest contained a median CFU-GM count of 45 x 10(4) kg-1 and 2 x 10(6) kg-1 CD34+ cells. Treatment with filgrastim (5 micrograms kg-1) following cyclophosphamide (3 g m-2) resulted in a harvest containing 66 x 10(4) kg-1 CFU-GM and 2.4 x 10(6) kg-1 CD34+ cells. There was no statistically significant difference between these two mobilising regimens. We have also demonstrated that dose-intensive carboplatin and cyclophosphamide chemotherapy can be delivered safely to patients with ovarian cancer when supported by peripheral blood progenitor cells and filgrastim. Carboplatin (AUC 7.5) and cyclophosphamide (900 mg m-2) given at 3 weekly intervals with progenitor cell and growth factor support was well tolerated in terms of haematological and systemic side-effects. Double the dose intensity of chemotherapy was delivered compared with our standard dose regimen when the treatment was given at 3 weekly intervals. Median dose intensity could be further escalated to 2.33 compared with our standard regimen by decreasing the interval between treatment cycles to 2 weeks. However, at this dose intensity less than a third of patients received their planned treatment on time. All the delays were due to thrombocytopenia.
    • Study of the proliferation in human gastric mucosa after in vivo bromodeoxyuridine labelling

      Patel, S; Rew, D A; Taylor, I; Potten, Christopher S; Owen, C; Roberts, Stephen A; The University Surgical Unit, Southampton General Hospital, Southampton (1993)
    • A study of the template properties of chromatin for DNA polymerase I and of the effects of ionising radiation.

      Itzhaki, Ruth F; Saffhill, Roy; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1973-06)
    • Study protocol for the DETECTIVE study: an international collaborative study to develop consensus statements for deferred treatment with curative intent for localised prostate cancer.

      Lam, TBL; MacLennan, S; Plass, K; Willemse, PM; Mason, MD; Cornford, P; Donaldson, J; Davis, NF; Dell'Oglio, P; Fankhauser, C; et al. (2018)
    • STUDY15: a multicentre, randomised trial comparing combination gemcitabine/carboplatin and hydroxychloroquine versus carboplatin/etoposide therapy for stage IV small-cell lung cancer (SCLC).

      Ngai, Y; Hackshaw, A; Blackhall, Fiona H; Greystoke, A; Ahmed, S; El-Khouly, F; Farrelly, L; Jenner, R; Bremner, F; Salomoni, P; et al. (2018-01)
    • Studying catabolism of protein ADP-Ribosylation.

      Palazzo, L; James, Dominic I; Waddell, Ian D; Ahel, I; Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK (2017)
      Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD(+)) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.
    • Subclone eradication analysis identifies targets for enhanced cancer therapy and reveals L1 retrotransposition as a dynamic source of cancer heterogeneity

      Ketola, K.; Kaljunen, H.; Taavitsainen, S.; Kaarijärvi, R.; Järvelä, E.; Rodriguez Martin, B.; Haase, K.; Woodcock, D. J.; Tubio, J.; Wedge, David C; et al. (2021)
      Treatment-eradicated cancer subclones have been reported in leukemia and have recently been detected in solid tumors. Here we introduce Differential Subclone Eradication and Resistance Analysis (DSER), a method developed to identify molecular targets for improved therapy by direct comparison of genomic features of eradicated and resistant subclones in pre- and post-treatment samples from a patient with BRCA2-deficient metastatic prostate cancer. FANCI and EYA4 were identified as candidate DNA repair-related targets for converting subclones from resistant to eradicable, and RNAi-mediated depletion of FANCI confirmed it as a potential target. The EYA4 alteration was associated with adjacent L1 transposon insertion during cancer evolution upon treatment, raising questions surrounding the role of therapy in L1 activation. Both carboplatin and enzalutamide turned on L1 transposon machinery in LNCaP and VCaP but not in PC-3 and 22Rv1 prostate cancer cell lines. L1 activation in LNCaP and VCaP was inhibited by the antiretroviral drug azidothymidine. L1 activation was also detected post-castration in LuCaP 77 and LuCaP 105 xenograft models and post-chemotherapy in previously published time-series transcriptomic data from SCC25 head and neck cancer cells. In conclusion DSER provides an informative intermediate step toward effective precision cancer medicine and should be tested in future studies, especially those including dramatic but temporary metastatic tumor regression. L1 transposon activation may be a modifiable source of cancer genomic heterogeneity, suggesting the potential of leveraging newly discovered triggers and blockers of L1 activity to overcome therapy resistance.
    • Subpathologies and genomic classifier for treatment individualization of post-prostatectomy radiotherapy

      Ramotar, M.; Chua, M. L. K.; Truong, H.; Hosni, A.; Pintilie, M.; Davicioni, E.; Fleshner, N. E.; Dicker, A. P.; Bristow, Robert G; He, H. H.; et al. (2021)
      Purpose/objective: Risk-stratification for post-prostatectomy radiotherapy (PORT) using conventional clinicopathologic indexes leads to substantial over- and under-treatment. Better patient selection could spare unnecessary toxicities and improve outcomes. We investigated the prognostic utility of unfavorable subpathologies intraductal carcinoma and cribriform architecture (IDC/CA), and a 22-gene Decipher genomic classifier (GC) in prostate cancer (PCa) patients receiving PORT. Material/methods: A cohort of 302 men who received PORT at 2 academic institutions was pooled. PORT was predominately delivered as salvage (62% of cases); 20% received HT+PORT. Specimens were centrally reviewed for IDC/CA presence. In 104 cases, GC scores were determined. Endpoints were biochemical relapse-free (bRFR) and metastasis-free (mFR) rates. Results: After a median follow-up of 6.49-years, 135 (45%) and 40 (13%) men experienced biochemical relapse and metastasis, respectively. IDC/CA were identified in 160 (53%) of cases. Men harboring IDC/CA experienced inferior bRFR (HR 2.6, 95%CI 1.8-3.2, P<0.001) and mFR (HR 3.1, 95%CI 1.5-6.4, P = 0.0014). Patients with GC scores, 22 (21%) were stratified low-, 30 (29%) intermediate-, and 52 (50%) high-risk. GC low-risk was associated with superior bRFR (HR 0.25, 95%CI 0.1-0.5, P<0.001) and mFR (HR 0.15, 95%CI 0.03-0.8, P = 0.025). On multivariable analyses, IDC/CA and GC independently predicted for bRFR, corresponding to improved discrimination (C-index = 0.737 (95%CI 0.662-0.813)). Conclusions: IDC/CA subpathologies and GC predict for biochemical relapse and metastasis beyond conventional clinicopathologic indexes in the PORT setting. Patients harboring IDC/CA are at higher risk of relapse after maximal local therapies, thus warranting consideration for treatment intensification strategies. Conversely, for men with absence of IDC/CA and low GC scores, de-intensification strategies could be explored.
    • Substituting angiotensin-(1-7) to prevent lung damage in SARSCoV2 infection?

      Peiro, C; Moncada, Salvador; Department of Pharmacology, School of Medicine, Universidad Autonoma de Madrid, Madrid, Spain (2020)
    • A Subtype of Olfactory Bulb Interneurons Is Required for Odor Detection and Discrimination Behaviors.

      Takahashi, H; Ogawa, Y; Yoshihara, S; Asahina, R; Kinoshita, M; Kitano, T; Kitsuki, M; Tatsumi, K; Okuda, M; Tatsumi, K; et al. (2016-08-03)
      Neural circuits that undergo reorganization by newborn interneurons in the olfactory bulb (OB) are necessary for odor detection and discrimination, olfactory memory, and innate olfactory responses, including predator avoidance and sexual behaviors. The OB possesses many interneurons, including various types of granule cells (GCs); however, the contribution that each type of interneuron makes to olfactory behavioral control remains unknown. Here, we investigated the in vivo functional role of oncofetal trophoblast glycoprotein 5T4, a regulator for dendritic arborization of 5T4-expressing GCs (5T4 GCs), the level of which is reduced in the OB of 5T4 knock-out (KO) mice. Electrophysiological recordings with acute OB slices indicated that external tufted cells (ETCs) can be divided into two types, bursting and nonbursting. Optogenetic stimulation of 5T4 GCs revealed their connection to both bursting and nonbursting ETCs, as well as to mitral cells (MCs). Interestingly, nonbursting ETCs received fewer inhibitory inputs from GCs in 5T4 KO mice than from those in wild-type (WT) mice, whereas bursting ETCs and MCs received similar inputs in both mice. Furthermore, 5T4 GCs received significantly fewer excitatory inputs in 5T4 KO mice. Remarkably, in olfactory behavior tests, 5T4 KO mice had higher odor detection thresholds than the WT, as well as defects in odor discrimination learning. Therefore, the loss of 5T4 attenuates inhibitory inputs from 5T4 GCs to nonbursting ETCs and excitatory inputs to 5T4 GCs, contributing to disturbances in olfactory behavior. Our novel findings suggest that, among the various types of OB interneurons, the 5T4 GC subtype is required for odor detection and discrimination behaviors.
    • Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis

      Carrol, Enitan D; Salway, Fiona; Pepper, Stuart D; Saunders, Emma K; Mankhambo, Limangeni A; Ollier, William E; Hart, C Anthony; Day, Phillip; Malawi-Liverpool-Wellcome Trust Clinical Research Programme, PO Box 30096, Blantyre, Malawi, Africa. edcarrol@liv.ac.uk (2007)
      BACKGROUND: The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use.The aim of this study was to modify the PAXgene Blood RNA System kit protocol for application to small, sick children, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression.Aliquots of 0.86 mL PAXgene reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease. RESULTS: Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02). CONCLUSION: We have successfully modified the PAXgene blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis.
    • Successive addition of electrons to sodium quinizarin-2- and -6-sulphonate in aqueous solution. a pulse and -radiolysis study

      Mukherjee, Tulsi; Land, Edward J; Swallow, A John; Guyan, P M; Bruce, J M (1988)
      Absorption characteristics of the semiquinone free radicals formed by one-electron reduction of quinizarin 2- and 6-sulphonates (Q2S and Q6S, respectively) have been studied. Second-order rate constants have been determined for the reactions of e, CO, H˙ and O with the two quinones and also for the reaction of the semiquinones with O2 and with themselves. The one-electron reduction potentials (vs. NHE) are E=–270 mV for Q2S and –249 mV for Q6S. They vary with pH in accordance with the pKa values of the parent and the semiquinone. The radicals are stable within the pH range 5–11. The stability constant is highest at pH 8.5, viz. ca. 1.45 for Q2S and 0.95 for Q6S. The one-electron reduction potentials of the semiquinones and the two electron reduction potentials of the quinones can be calculated to be E=–246 mV for Q2S and –213 mV for Q6S, and E=–258 mV for Q2S and –231 mV for Q6S. The variation of these quantities with pH has been related to pKa values for the relevant species. Properties of the radiolytically prepared fully reduced Q2S and Q6S are reported. Their acid forms are stable to oxygen. Adjustment to alkaline pH results in a loss of the sulphonate group from fully reduced Q2S but not from fully reduced Q6S. Reasons are given.
    • Sudan Black B, the specific histochemical stain for lipofuscin: a novel method to detect senescent cells.

      Evangelou, K; Gorgoulis, Vassilis G; Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, Mikras Asias 75, Athens, 11527, Greece (2017)
      The Sudan-Black-B (SBB) histochemical stain is well known to specifically react against lipofuscin, an aggregate of oxidized proteins, lipids, and metals. Lipofuscin is related to many ageing processes. It is also known to accumulate in senescent cells. We recently proved that lipofuscin detection, when applying the SBB staining, is highly specific for the visualization of senescent cells. Here, we present in detail this SBB method that can detect senescent cells in any material, irrespective of its preparation. This provides unique advantages not only in understanding physiological processes and the pathophysiology of various diseases but also in estimating the response to therapeutic interventions.