Anti-Inflammatory Diet for Inflammatory Bowel Disease (IBD-AID)

Background: Inflammatory Bowel Disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic, immune-mediated inflammatory conditions of the gastrointestinal tract, which have increasingly been linked to dysbiosis, or an imbalance in the gut microbiome. Standard of care for IBD involves an often-evolving combination of anti-inflammatory, antibiotic, and immunomodulatory medications; however, the pharmacological approach is never curative, and medications routinely become ineffective for individual patients. Partially fueled by the increasing inadequacy of pharmacologic treatment regimens, there is emerging interest from patients regarding diet and its role in the pathogenesis and treatment of inflammatory diseases, demanding more in-depth and substantiating research from the medical community. The Anti-Inflammatory Diet for IBD (IBD-AID), which is derived and augmented from The Specific Carbohydrate Diet (SCD), is a nutritional regimen that restricts the intake of pro-inflammatory carbohydrates such as refined sugar, lactose, and most grains, while maximizing anti-inflammatory foods including those with prebiotic and probiotic properties. We have previous results from a case series of 11 patients with IBD showing symptomatic improvement (by Harvey Bradshaw Index scores) and downscaling of medication regimens in all 11 patients after 4 weeks on the IBD-AID. Objectives: The purpose of this small prospective study was to further assess the efficacy and feasibility of the IBD-AID intervention for the treatment of CD, and to provide pilot data for a larger application. Methods: The sample included 17 patients with biopsy-confirmed Crohn’s disease. Participants were offered the treatment diet (IBD-AID) (n=12) or standard medical care alone (control) (n=5). Patients in the IBD-AID group were required to attend one individual nutrition counseling session and three IBD-AID-specific cooking classes at the University of Massachusetts Medical School’s Shaw Building teaching kitchen. The control group continued with usual care. For all participants, demographic, clinical, and symptom data were obtained from baseline and follow-up questionnaires; dietary composition was monitored by weekly dietary recalls and food journals. All participants continued to follow with their gastroenterologists throughout the study duration. Study duration was 2 months after 70% adherence to the diet for IBD-AID participants, and 2 months after baseline for control participants. Consistent with the goals for any treatment used for CD, efficacy measures included: 1) reduction in symptomology, as measured by the validated Harvey Bradshaw Index (HBI); 2) reduction in the need of immunomodulatory and anti-inflammatory medications; and 3) normalizing trend in circulating inflammatory markers (i.e., CRP and ESR), albumin, and hematocrit. Feasibility measures included participant retention, dietary compliance, and participants’ self-assessments of difficulty in maintaining the diet. Results: A total of 15 enrolled patients with confirmed diagnosis of Crohn’s Disease, 5 in observation arm, 10 in intervention arm. Significant trends in dietary composition included significant increases in prebiotic and favorable dietary components, and decrease in adverse foods for the group as a whole (paired t-test values 0.0016, 0.0344, 0.0085, and 0.0014, respectively). For those patients on medication at baseline and with complete follow-up (n=9), one-third were able to decrease doses of or discontinue these medications. In addition, lab values reflected symptomatic improvements in two of our intervention patients, with changes in CRP, ESR, and hematocrit levels of -55.9 and -1.4, -30.0 and -15.0, and +5.4 and +0.3, respectively, with corresponding symptomatic improvements (HBI scores 1à7 and 8à0, respectively). No significance can be assigned, however, due to low sample size and loss to follow-up. Feasibility measures include a significant loss to follow-up rate of 33.3%, as well as an average “difficulty score” of 3.1, reflecting participants’ views on the difficult nature of “sticking with” the IBD-AID (scored on scale of 1-5, very easy to very difficult). Conclusion: Despite the study’s limitations, as well as because of them, several conclusions can be drawn. The trends noticed in the participants’ dietary component reports, and supported by participants’ self-evaluation, reveal that it is relatively easy to eliminate problem foods from the diet, but adding unfamiliar foods, particularly from the probiotic category such as plain yogurt, kimchi, miso, sauerkraut, etc., is a huge barrier to maintaining compliance. This trend may be a partial reflection of the Western food and dieting culture in which our daily meals are relatively homogenous. We are also brought up from a young age learning that “dieting” and “healthy eating” means cutting out the bad, but not necessarily bringing in the good and/or new. Despite lack of statistical significance, the two patients who exhibited normalizing lab values, in combination with their improved HBI scores, suggest the possibility of a real and meaningful benefit from IBD-AID for those able to comply with the dietary and lifestyle changes. In terms of the diet’s feasibility, the considerable loss to follow-up in this study may reflect a variety of issues, one of which may be the well-established medical and psychosocial complexity of IBD patients. This element is important for clinicians to keep in mind, and reflects the need for additional support and close follow-up when it comes to facilitating lifestyle change in this population. It also has implications for the diet itself, which should be re-examined to simplify or reframe in order to maximize generalizability and access for a greater percentage of IBD patients. Overall, this small study highlights the need for larger-scale research to draft clinical nutrition guidelines and further legitimize the utility of preventive clinical nutrition in Western medicine

In vitro prediction of polycyclic aromatic hydrocarbon bioavailability of 14 different incidentally ingested soils in juvenile swine

Predicting mammalian bioavailability of PAH mixtures from in vitro bioaccessibility results has proven to be an elusive goal. In an attempt to improve in vitro predictions of PAH soil bioavailability we investigated how energetic input influences PAH bioaccessibility by using a high and low energetic shaking method. Co-inertia analysis (COIA), and Structural Equation Modeling (SEM) were also used to examine PAH-PAH interactions during ingestion. PAH bioaccessibility was determined from 14 historically contaminated soils using the fed organic estimation of the human simulation test (FOREhST) with inclusion of a silicone rod as a sorption sink and compared to bioavailability estimates from the juvenile swine model. Shaking method significantly affected PAH bioaccessibility in the FOREhST model, with PAH desorption from the high energy FOREhST almost an order of magnitude greater compared to the low energy FOREhST. PAH-PAH interactions significantly influenced PAH bioavailability and when these interactions were used in a linear model, the model predicted benzo(a)anthracene bioavailability with an slope of 1 and r2 of 0.66 and for benzo(a)pyrene bioavailability has a slope of 1 and r2 of 0.65. Lastly, to confirm the effects as determined by COIA and SEM, we spiked low levels of benzo(a)anthracene into historically contaminated soils, and observed a significant increase in benzo(a)pyrene bioaccessibility. By accounting for PAH interactions, and reducing the energetics of in vitro extractions, we were able to use bioaccessibility to predict bioavailability across 14 historically contaminated soils. Our work suggests that future work on PAH bioavailability and bioaccessibility should focus on the dynamics of how the matrix of PAHs present in the soil interact with mammalian systems. Such interactions should not only include the chemical interactions discussed here but also the interactions of PAH mixtures with mammalian uptake systems

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The evolution of lung cancer and impact of subclonal selection in TRACERx

Lung cancer is the leading cause of cancer-associated mortality worldwide. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource

The evolution of non-small cell lung cancer metastases in TRACERx

Metastatic disease is responsible for the majority of cancer-related deaths. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse

Genomic–transcriptomic evolution in lung cancer and metastasis

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic–transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary–metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS). Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response

A Geochemical Study of Hydrothermal Signals in Marine Sediments: The Rainbow Hydro thermal Area, 36 degrees on the Mid-Atlantic Ridge

The geochemistry of a suite of cores accumulating at increasing distance down-stream of a major hydrothermal vent site has been characterized for the first time, in order to examine the changing chemistry of particles being deposited from the plume with distance from the vent, to try to establish the different processes operating in the plume, particularly with regard to scavenging of elements from seawater, and to quantify the fluxes from seawater to the sediments as a result of venting, for a wide range of major and trace elements (Mg, Fe, V, Mn, Co, Cu, Zn, REE, PGE, Os isotopes).Sediment cores were collected at 2-25km downplume of the Rainbow hydrothermal vent site at 36o14'N on the Mid-Atlantic Ridge, a known site of vigorous high-temperature venting. Samples from long-term sediment traps located 0.5-2 km from this vent were also studied. All samples showed similar bulk chemistry, with an overprint of hydrothermally derived material. Sedimentation rates of 40-60 mg.m2d-1 for the cores were established using bulk radiocarbon dating of the carbonate material, and were found to be of the same order as the sediment traps 11-25 mg.m2.d-1. The maximum age of the core samples ranged from 8-26 x 103 radiocarbon years.For many elements, particularly the PGEs, the element to iron ratios in plume derived material were found to be up to an order of magnitude higher than those found globally in metalliferous sediments of hydrogenous origin. Burial fluxes to the Rainbow sediments have been calculated for hydrothermally derived elements - Mn (16-37 &mu;mol.cm-2.kyr-1), Fe (368-718&mu;mol.cm-2.kyr-1), Co(0.3-0.5&mu;mol.cm-2.kyr-1), Cu(3.5-12&mu;mol.cm-2.kyr-1), Zn(0.2-0.6&mu;mol.cm-2.kyr-1), - and elements scavenged from seawater by hydrothermal Fe-oxyhydroxides - P (36-64&mu;mol.cm-2.kyr-1), V (3-5&mu;mol.cm-2.kyr-1), As (0.6-1.4&mu;mol.cm-2.kyr-1), Pd(20-53pmol.cm-2.kyr-1), Os (0.1-2.3pmol.cm-2.kyr-1), Ir(0.2-0.6pmol.cm-2.kyr-1), Pt(6.5 - 18.5pmol.cm-2.kyr-1). Magnesium from seawater appears to co-precipitate with Fe in the vent plume at this site, and Mg burial fluxes are calculated to be 73-146&mu;mol.cm-2.kyr-1 . Burial fluxes of the hydrothermally derived elements represent only a fraction of the estimated vent output, implying that vent products are dispersed over long distances. Element/Fe ratios for scavenged elements are high compared to other metalliferous sediments, indicating enhanced scavenging efficiency at this site.</p