The dysfunctional immune response in renal cell carcinoma correlates with changes in the metabolic landscape of ccRCC during disease progression.

Renal cell carcinoma is an immunogenic tumour with a prominent dysfunctional immune cell infiltrate, unable to control tumour growth. Although tyrosine kinase inhibitors and immunotherapy have improved the outlook for some patients, many individuals are non-responders or relapse despite treatment. The hostile metabolic environment in RCC affects the ability of T-cells to maintain their own metabolic programme constraining T-cell immunity in RCC. We investigated the phenotype, function and metabolic capability of RCC TILs correlating this with clinicopathological features of the tumour and metabolic environment at the different disease stages. Flow cytometric analysis of freshly isolated TILs showed the emergence of exhausted T-cells in advanced disease based on their PD-1high and CD39 expression and reduced production of inflammatory cytokines upon in vitro stimulation. Exhausted T-cells from advanced stage disease also displayed an overall phenotype of metabolic insufficiency, characterized by mitochondrial alterations and defects in glucose uptake. Nanostring nCounter cancer metabolism assay on RNA obtained from 30 ccRCC cases revealed significant over-expression of metabolic genes even at early stage disease (pT1-2), while at pT3-4 and the locally advanced thrombi stages, there was an overall decrease in differentially expressed metabolic genes. Notably, the gene PPARGC1A was the most significantly down-regulated gene from pT1-2 to pT3-4 RCC which correlated with loss of mitochondrial function in tumour-infiltrating T-cells evident at this tumour stage. Down-regulation of PPARGC1A into stage pT3-4 may be the 'tipping-point' in RCC disease progression, modulating immune activity in ccRCC and potentially reducing the efficacy of immunotherapies in RCC and poorer patient outcomes.

Financial toxicity in lung cancer: an assessment of magnitude, perception, and impact on quality of life.

BACKGROUND: Advances in lung cancer therapy have resulted in improved clinical outcomes. Unfortunately, advances can come at a financial cost to patients and their families that poses a significant risk to overall quality of life (QoL). Financial distress has been shown to be associated with increased symptom burden and decreased treatment compliance but the magnitude of financial distress is not well characterized in lung cancer populations. PATIENTS AND METHODS: Patients with stage II-IV newly diagnosed lung cancer and starting first-line therapy were recruited at a tertiary academic institution between July 2018 and April 2019. The comprehensive score for financial toxicity (COST) was used to assess financial toxicity and the Functional Assessment of Cancer Therapy-Lung (FACT-L) was used to assess QoL. Associations between financial toxicity and baseline variables were assessed using multivariable linear regression and correlations were assessed using the Pearson correlation. RESULTS: In this study, 143 consecutive patients were approached and 91.6% agreed to participate (N = 131). The median age was 65 years (35-90); 52.7% were male (n = 69), and 75.6% were white (n = 99). The inability to afford basic necessities and having <1 month of savings was associated with increased financial toxicity (P < 0.001) after adjusting for other factors such as age, race, insurance, and income. There was also a trend toward increased financial toxicity among those who were employed but on sick leave (P = 0.06). Increased financial toxicity was correlated with a decrease in QoL (correlation coefficient 0.41, P < 0.001). Patients' anticipated out-of-pocket (OOP) expenses for the upcoming 6 months ranged from $0 to $50 000 (median $2150). However, there was no correlation between anticipated OOP expenses and either financial toxicity or QoL. CONCLUSIONS: These data identify key factors for identifying at-risk patients and builds a framework for exploring the benefit of financial counseling interventions, which may improve QoL and oncologic outcomes.

Influencing surgical management in patients with carcinoma of the cervix using a T2- and ZOOM-diffusion-weighted endovaginal MRI technique.

BACKGROUND: Endovaginal MRI (evMRI) at 3.0-T with T2-weighted (T2-W) and ZOnal Oblique Multislice (ZOOM)-diffusion-weighted imaging (DWI) potentially improves the detection of stage Ia/Ib1 cervical cancer. We aimed to determine its sensitivity/specificity, document tumour-to-stromal contrast and establish the effect of imaging on surgical management. METHODS: Following ethical approval and written informed consent, 57 consecutive patients with suspected stage Ia/Ib1 cervical cancer underwent evMRI at 3.0-T using T2-W and ZOOM-DWI. Sensitivity/specificity were calculated against histopathology for two independent observers. Tumour-to-stromal contrast was determined on T2-W, and diffusion-weighted (b=800 s mm(-2)) images and apparent diffusion coefficients (ADCs) were recorded. In patients due for radical vaginal trachelectomy (RVT), change of surgical management based on imaging findings was documented. RESULTS: Sensitivity/specificity for detecting tumour was the following: reporting read 88.0/81.8%, anonymised read 92.0/81.8% (observer 1); 84.0/72.7% (observer2; median tumour volume=1.7 cm(3)). Intraobserver agreement was excellent (kappa=0.89) and the interobserver agreement was good (kappa=0.65). Tumour-to-stromal contrast was greater on ZOOM-DWI compared with T2-W images (3.35±2.36 vs 1.39±0.95; P<0.0004). Tumour and stromal ADCs were significantly different (P<0.00001). In 31 patients due for RVT, evMRI altered surgical management in 12 (38.7%) cases (10 cone-biopsy, 2 chemoradiotherapy). CONCLUSION: T2-W+ZOOM-DWI evMRI has high sensitivity/specificity for detecting stage Ia/Ib1 cervical tumours; in patients due for RVT, the surgical management was altered in ∼39%.

Validation of T2- and diffusion-weighted magnetic resonance imaging for mapping intra-prostatic tumour prior to focal boost dose-escalation using intensity-modulated radiotherapy (IMRT).

BACKGROUND AND PURPOSE: To assess the diagnostic accuracy and inter-observer agreement of T2-weighted (T2W) and diffusion-weighted (DW) magnetic resonance imaging (MRI) for mapping intra-prostatic tumour lesions (IPLs) for the purpose of focal dose-escalation in prostate cancer radiotherapy. MATERIALS AND METHODS: Twenty-six men selected for radical treatment with radiotherapy were recruited prospectively and underwent pre-treatment T2W+DW-MRI and 5 mm spaced transperineal template-guided mapping prostate biopsies (TTMPB). A 'traffic-light' system was used to score both data sets. Radiologically suspicious lesions measuring ≥0.5 cm3 were classified as red; suspicious lesions 0.2-0.5 cm3 or larger lesions equivocal for tumour were classified as amber. The histopathology assessment combined pathological grade and tumour length on biopsy (red = ≥4 mm primary Gleason grade 4/5 or ≥6 mm primary Gleason grade 3). Two radiologists assessed the MRI data and inter-observer agreement was measured with Cohens' Kappa co-efficient. RESULTS: Twenty-five of 26 men had red image-defined IPLs by both readers, 24 had red pathology-defined lesions. There was a good correlation between lesions ≥0.5 cm3 classified "red" on imaging and "red" histopathology in biopsies (Reader 1: r = 0.61, p < 0.0001, Reader 2: r = 0.44, p = 0.03). Diagnostic accuracy for both readers for red image-defined lesions was sensitivity 85-86%, specificity 93-98%, positive predictive value (PPV) 79-92% and negative predictive value (NPV) 96%. Inter-observer agreement was good (Cohen's Kappa 0.61). CONCLUSIONS: MRI is accurate for mapping clinically significant prostate cancer; diffusion-restricted lesions ≥0.5 cm3 can be confidently identified for radiation dose boosting.

Metabolism and genetics of Helicobacter pylori: the genome era.

The publication of the complete sequence of Helicobacter pylori 26695 in 1997 and more recently that of strain J99 has provided new insight into the biology of this organism. In this review, we attempt to analyze and interpret the information provided by sequence annotations and to compare these data with those provided by experimental analyses. After a brief description of the general features of the genomes of the two sequenced strains, the principal metabolic pathways are analyzed. In particular, the enzymes encoded by H. pylori involved in fermentative and oxidative metabolism, lipopolysaccharide biosynthesis, nucleotide biosynthesis, aerobic and anaerobic respiration, and iron and nitrogen assimilation are described, and the areas of controversy between the experimental data and those provided by the sequence annotation are discussed. The role of urease, particularly in pH homeostasis, and other specialized mechanisms developed by the bacterium to maintain its internal pH are also considered. The replicational, transcriptional, and translational apparatuses are reviewed, as is the regulatory network. The numerous findings on the metabolism of the bacteria and the paucity of gene expression regulation systems are indicative of the high level of adaptation to the human gastric environment. Arguments in favor of the diversity of H. pylori and molecular data reflecting possible mechanisms involved in this diversity are presented. Finally, we compare the numerous experimental data on the colonization factors and those provided from the genome sequence annotation, in particular for genes involved in motility and adherence of the bacterium to the gastric tissue.

Characterisation of glucose transport in Helicobacter pylori.

The nature of the glucose transport system in the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis. Fast D-glucose uptake was demonstrated by using two methods of measuring glucose transport. The transport of 2-deoxy-D-glucose was inhibited competitively by D-glucose; and the efflux of 2-deoxy-D-glucose from cells also was affected by the presence of D-glucose. The transport of 2-deoxy-D-glucose was saturable with a Km of 4.8 mM and Vmax of 146.6 pmol (microliter cell water)-1 at 20 degrees C. The transport was temperature-dependent with energies of activation of 6.8 and 51.0 kJ mol-1 for 0.2 and 20 mM 2-deoxy-D-glucose, respectively. The temperature dependence and saturable nature of transport suggested the presence of one or more glucose carriers. The substrate specificity of the transport system was studied by measuring the effects of mono- and disaccharides on the rates of transport of the glucose analogue. The most significant inhibitory effects were obtained with D-galactose and L-arabinose. Lack of transport inhibition by L-glucose established the stereospecificity of the transporters for the D-isomer of glucose. Higher rates of 2-deoxy-D-glucose transport were measured in the presence of sodium ions than for other monovalent cations, and the presence of amiloride inhibited transport of the monosaccharide. No inhibition was observed with cytochalasin B, phloretin or phloridzin. The results suggested the existence of specific D-glucose transporters and that the glucose transport system of H. pylori is significantly different from other known bacterial transporters.

Aminoacid utilization by Helicobacter pylori.

Utilization of aminoacids during growth by laboratory adapted and wild type Helicobacter pylori strains was investigated employing nuclear magnetic resonance spectroscopy and aminoacid analysis. All H. pylori strains tested showed growth rates with doubling times of approx. 11.5 hr in liquid cultures with semi-defined media or with defined aminoacid broth without carbohydrates. Fast utilization of several aminoacids at rates between 80 and 250 microM/hr was observed in culture broths inoculated with approx. 10(7) cells/ml; and acetate, formate and succinate accumulated as catabolic products in the growth media. Suspensions of bacterial cells and lysates in isotonic solutions converted arginine, asparagine, aspartate, glutamine, and serine used as sole substrates at significant rates; and under these conditions the principal metabolic products observed were acetate, formate, succinate and lactate. The findings of the study indicated that H. pylori can survive employing aminoacids as the basic nutrients, and suggested some of these metabolites were utilized via fermentative pathways with common characteristics to those found in anaerobes.

Oxidases and reductases are involved in metronidazole sensitivity in Helicobacter pylori.

Helicobacter pylori is a contributing factor to the development of gastric and duodenal ulcers and some gastric cancers. Some therapeutic regimes comprise of a number of components, one of which is the antimicrobial metronidazole. A problem with these therapies is the increasing prevalence of metronidazole-resistant (MtrR) H. pylori strains. Several resistance mechanisms have been proposed, and this study addresses the 'scavenging of oxygen' hypothesis. Spectrophotometric assays of cytosolic fractions indicated that metronidazole-sensitive (MtrS) H. pylori isolates had 2.6-fold greater nicotinamide adenine dinucleotide (NADH) oxidase activity, 34-fold greater NADH nitroreductase activity, and eightfold greater nicotinamide adenine dinucleotide phosphate (NADPH) nitroreductase activity than cytosolic fractions from matched MtrR strains. Electrophoresis of cytosolic fractions in non-denaturing gels showed up to 10 protein bands when stained with Coomassie blue. Activity staining of non-denaturing, non-reducing polyacrylamide gels detected NAD(P)H oxidase, disulphide reductase, tetrazolium reductase and nitroreductase activities in the protein bands. Oxidase and reductase activities observed in a band from MtrS strains were absent in the corresponding band from MtrR strains. This band comprised at least 13 proteins, and the major constituent was identified as an alkyl hydroperoxide reductase AhpC subunit. The absence of oxidase and reductase activities in the band from MtrR strains indicated a correlation between the activity of the proteins in this band and the metronidazole-sensitive phenotype.

Fumarate metabolism and the microaerophily of Campylobacter species.

(1) The role of fumarate metabolism in the microaerophily of the Campylobacter genus and the effects of therapeutic agents against it were investigated. (2) NMR spectroscopy was employed to determine the properties of Campylobacter fumarase (Fum) and fumarate reductase (Frd). Radiotracer analysis was used to determine the production of carbon dioxide by Campylobacter cells. Standard microbiological techniques were used to measure the effects of environmental conditions and inhibitors on bacterial growth. (3) All Campylobacter species tested showed both Fum and Frd activities. Frd activity was observed with or without the addition of an exogenous electron donor in the particulate fractions obtained from lysates. Fumarate was oxidized to carbon dioxide via the acetyl-CoA cleavage pathway. The genes encoding proteins involved in fumarate metabolism were identified in the Campylobacter jejuni genome. Cells grew better in atmospheres with 5 and 10% oxygen levels. Fum activity was the same in cultures grown under different oxygen tensions and did not vary with the age of cultures. Frd activity was higher in cultures which grew at faster rates and decreased with the age of cultures. Four Frd inhibitors showed bactericidal effects against Campylobacter spp. with different potencies. The relative strengths of inhibition of the compounds followed the same order as the bactericidal effects. (4) The results suggested that Frd and Fum are constitutive and play a fundamental role in these microaerophiles which show characteristics of anaerobic metabolism, and that the Frd inhibitors tested would not be of therapeutic use.

Genetic relationship among isolates of Helicobacter pylori: evidence for the existence of a Helicobacter pylori species-complex.

We investigated the population genetics of 23 isolates of H. pylori by allozyme electrophoresis using 16 enzyme loci. Isolates were obtained from adult patients of whom 48% were of Greek extraction. Eight patients (35%) had an active duodenal ulcer. Allelic variation per loci ranged from 2 to 11 alleles. Four major genetic clusters were apparent, having > 75% fixed genetic differences. There was no distinct clustering (clonal structure) on the basis of the geographical origin of the persons from whom isolates were obtained, indicating that this bacterium has not recently jumped a species barrier into humans. Isolates associated with ulcer disease were not monophyletic, with isolates from ulcer patients being found in phylogenetically diverse branches of the dendogram derived from the data. Based on the genetic diversity of H. pylori isolates, we propose that isolates should be classified as belonging not to a single species but to a 'Helicobacter pylori species-complex'.

Lack of protection against gastric Helicobacter infection following immunisation with jack bean urease: the rejection of a novel hypothesis.

The common mucosal immune system was stimulated by oral immunisation with jack bean urease and the adjuvant cholera toxin. A high level of local antibody and serum antibody was induced in mice following hyperimmunisation with this combination. No cross-reacting antibody was found against either Helicobacter pylori or Helicobacter felis. No protection was observed against oral challenge of immunised mice with living H. felis thus disproving the interesting hypothesis of Pallen and Clayton that plant urease might induce a protective immunity against helicobacter infection.

The urease enzymes of Campylobacter pylori and a related bacterium.

The urease enzyme of Campylobacter pylori was studied and compared with that of a related spiral-shaped bacterium, St1, isolated from the rodent ileum. Both bacteria possessed constitutive urease enzymes with activities up to 20-70 times that of Proteus vulgaris. This activity was retained on SDS-polyacrylamide gels. A major catalytic subunit of mol. wt 300,000 was located for all (six) strains of C. pylori subjected to SDS-PAGE whereas St1 had two active forms of mol. wts 140,000 and 150,000. Western-blot analysis indicated the presence of anti-urease antibodies in the sera of patients with C. pylori-associated gastritis. The response to C. pylori urease was not strain-specific but no cross-reactivity was detected between the C. pylori enzyme and that of St1. The very high urease activity of these bacteria is likely to be important in colonisation of the host. Possession of glutamate dehydrogenase activity by both organisms suggests that one role of the urease may be to assimilate the available urea nitrogen. Modification of the local environment to facilitate long-term colonisation is another possible function. Protection from acid is unlikely to be a primary role as the natural habitat of the organism St1 is the non-acid-secreting tissue of the small intestine.

Time to hand over the baby--parental care of the preterm infant.

Parents of babies in neonatal units often feel distanced from their baby, saying there is nothing they can do to help. Staff in NNUs must help involve parents in the care of their baby, to soothe qualms about discharge.

Fumarate catabolism in Helicobacter pylori.

The metabolism of fumarate by Helicobacter pylori was investigated employing one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy. Metabolically competent cells generated malate and succinate from fumarate as the sole substrate indicating the presence of fumarase and fumarate reductase activities in the bacterium. In incubations of fumarate with cell lysates accumulation of lactate, acetate, formate and alanine was observed after the initial production of malate and succinate. The results indicate the existence of active fumarate catabolism in H. pylori and suggest the possibility of an ATP generating mechanism which may play an important role in the bioenergetics of the bacterium.