Chronic consequences of ischemic stroke: Profiling brain injury and inflammation in a mouse model with reperfusion.

Stroke is a pervasive and debilitating global health concern, necessitating innovative therapeutic strategies, especially during recovery. While existing literature often focuses on acute interventions, our study addresses the uniqueness of brain tissue during wound healing, emphasizing the chronic phase following the commonly used middle cerebral artery (MCA) occlusion model. Using clinically relevant endpoints in male and female mice such as magnetic resonance imaging (MRI) and plasma neurofilament light (NFL) measurement, along with immunohistochemistry, we describe injury evolution. Our findings document significant alterations in edema, tissue remodeling, and gadolinium leakage through MRI. Plasma NFL concentration remained elevated at 30 days poststroke. Microglia responses are confined to the region adjacent to the injury, rather than continued widespread activation, and boron-dipyrromethene (BODIPY) staining demonstrated the persistent presence of foam cells within the infarct. Additional immunohistochemistry highlighted sustained B and T lymphocyte presence in the poststroke brain. These observations underscore potentially pivotal roles played by chronic inflammation brought on by the lipid-rich brain environment, and chronic blood-brain barrier dysfunction, in the development of secondary neurodegeneration. This study sheds light on the enduring consequences of ischemic stroke in the most used rodent stroke model and provides valuable insights for future research, clinical strategies, and therapeutic development.

In vivo CAR T-cell generation in nonhuman primates using lentiviral vectors displaying a multidomain fusion ligand.

ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.

Real-world use of long-acting cabotegravir and rilpivirine: 12-month results of the inJectable Antiretroviral therapy feasiBility Study (JABS).

OBJECTIVES: The inJectable Antiretroviral feasiBility Study (JABS) aimed to evaluate the implementation of long-acting regimens in a 'real world' Australian setting, with inclusion of participants with complex medical needs, social vulnerability and/or historical non-adherence. METHODS: JABS was a 12-month, single-centre, single-arm, open-label phase IV study of long-acting cabotegravir 600 mg plus rilpivirine 900 mg administered intramuscularly every 2 months to adults with treated HIV-1 infection. The primary endpoint was the proportion of attendances and administration of injections within a 14-day dosing window over 12 months, out of the total prescribed doses. Secondary endpoints included proportions of missed appointments, use of oral bridging, discontinuations, virological failures, adverse events and participant-reported outcomes. A multidisciplinary adherence programme embedded in the clinical service known as REACH provided support to JABS participants. RESULTS: Of 60 participants enrolled by May 2022, 60% had one or more complexity or vulnerability factors identified, including absence of social supports (50%), mental health issues, alcohol or drug use (30%) and financial hardship or instability (13%), among others. Twenty-seven per cent of participants had historical non-adherence to antiretroviral therapy. Out of 395 prescribed doses, 97.2% of injections were administered within correct dosing windows at clinic visits. Two courses of short-term oral bridging were required. The rate of injection site reactions was 29%, the majority being grade 1-2. There were no virological failures, no serious adverse events and only one injection-related study discontinuation. High baseline treatment satisfaction and acceptability of injections increased by month 12. Those with vulnerability factors had similar adherence to injections as those without such factors. Ninety-eight per cent of the participants who completed 12 months on the study have maintained long-acting therapy, virological suppression and retention in care. CONCLUSIONS: Long-acting cabotegravir plus rilpivirine was associated with very high adherence, maintenance of virological suppression, safety and treatment satisfaction in a diverse Australian clinic population, comparable to results of phase III randomized clinical trials. Individuals with vulnerability factors can achieve adherence to injections with individualized support. Long-acting therapies in this group can increase the subsequent engagement in clinical care.

The Extra-Islet Pancreas Supports Autoimmunity in Human Type 1 Diabetes.

In autoimmune Type 1 diabetes (T1D), immune cells progressively infiltrate and destroy the islets of Langerhans - islands of endocrine tissue dispersed throughout the pancreas. However, it is unclear how this process, called 'insulitis', develops and progresses within this organ. Here, using highly multiplexed CO-Detection by indEXing (CODEX) tissue imaging and cadaveric pancreas samples from pre-T1D, T1D, and non-T1D donors, we examine pseudotemporal-spatial patterns of insulitis and exocrine inflammation within large pancreatic tissue sections. We identify four sub-states of insulitis characterized by CD8 + T cells at different stages of activation. We further find that exocrine compartments of pancreatic lobules affected by insulitis have distinct cellularity, suggesting that extra-islet factors may make particular lobules permissive to disease. Finally, we identify "staging areas" - immature tertiary lymphoid structures away from islets where CD8 + T cells appear to assemble before they navigate to islets. Together, these data implicate the extra-islet pancreas in autoimmune insulitis, greatly expanding the boundaries of T1D pathogenesis.

Single Circulating-Tumor-Cell-Targeted Sequencing to Identify Somatic Variants in Liquid Biopsies in Non-Small-Cell Lung Cancer Patients.

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths worldwide. Liquid biopsy by a blood draw to detect circulating tumor cells (CTCs) is a tool for molecular profiling of cancer using single-cell and next-generation sequencing (NGS) technologies. The aim of the study was to identify somatic variants in single CTCs isolated from NSCLC patients by targeted NGS. Thirty-one subjects (20 NSCLC patients, 11 smokers without cancer) were enrolled for blood draws (7.5 mL). CTCs were identified by immunofluorescence, individually retrieved, and DNA-extracted. Targeted NGS was performed to detect somatic variants (single-nucleotide variants (SNVs) and insertions/deletions (Indels)) across 65 oncogenes and tumor suppressor genes. Cancer-associated variants were classified using OncoKB database. NSCLC patients had significantly higher CTC counts than control smokers (p = 0.0132; Mann-Whitney test). Analyzing 23 CTCs and 13 white blood cells across seven patients revealed a total of 644 somatic variants that occurred in all CTCs within the same subject, ranging from 1 to 137 per patient. The highest number of variants detected in ≥1 CTC within a patient was 441. A total of 18/65 (27.7%) genes were highly mutated. Mutations with oncogenic impact were identified in functional domains of seven oncogenes/tumor suppressor genes (NF1, PTCH1, TP53, SMARCB1, SMAD4, KRAS, and ERBB2). Single CTC-targeted NGS detects heterogeneous and shared mutational signatures within and between NSCLC patients. CTC single-cell genomics have potential for integration in NSCLC precision oncology.

Beyond Circulating Tumor Cell Enumeration: Cell-Based Liquid Biopsy to Assess Protein Biomarkers and Cancer Genomics Using the RareCyte® Platform.

The practice of medicine has steadily employed less invasive methods to obtain information derived from the tumor to guide clinical management of patients. Liquid biopsy-the sampling of blood-is a non-invasive method for generating information previously only available from tissue biopsies of the tumor mass. Analysis of fragmented circulating tumor DNA in the plasma is clinically used to identify actionable mutations and detect residual or recurrent disease. Plasma analysis cannot, however, assess cancer phenotypes, including the expression of drug targets and protein biomarkers. Circulating tumor cells (CTCs) are intact cancer cells that have entered the blood that have the potential for distant metastasis. While enumeration of CTCs is prognostic of outcome, recently developed technology allows for the interrogation of protein biomarkers on CTCs that could be predictive of response. Furthermore, since CTCs contain intact whole cancer genomes, isolating viable CTCs detected during therapy could provide a rational approach to assessing mutational profiles of resistance. Identification, characterization and molecular analysis of CTCs together will advance the capacity of liquid biopsy to meet the requirements of twenty-first century medicine.

Identification and characterization of effusion tumor cells (ETCs) from remnant pleural effusion specimens.

BACKGROUND: Cancer is a leading cause of death worldwide, and patients may have advanced disease when diagnosed. Targeted therapies guided by molecular subtyping of cancer can benefit patients significantly. Pleural effusions are frequently observed in patients with metastatic cancer and are routinely removed for therapeutic purposes; however, effusion specimens have not been recognized as typical substrates for clinical molecular testing because of frequent low tumor cellularity. METHODS: Excess remnant pleural effusion samples (N = 25) from 21 patients with and without suspected malignancy were collected at Stanford Health Care between December 2019 and November 2020. Samples were processed into ThinPrep slides and underwent novel effusion tumor cell (ETC) analysis. The ETC results were compared with the original clinical diagnoses for accuracy. A subset of confirmed ETCs was further isolated and processed for molecular profiling to identify cancer driver mutations. All samples were obtained with Institutional Review Board approval. RESULTS: The authors established novel quantitative standards to identify ETCs and detected epithelial malignancy with 89.5% sensitivity and 100% specificity in the pleural effusion samples. Molecular profiling of confirmed ETCs (pools of 5 cells evaluated) revealed key pathogenic mutations consistent with clinical molecular findings. CONCLUSIONS: In this study, the authors developed a novel ETC-testing assay that detected epithelial malignancies in pleural effusions with high sensitivity and specificity. Molecular profiling of 5 ETCs showed promising concordance with the clinical molecular findings. To promote cancer subtyping and guide treatment, this ETC-testing assay will need to be validated in larger patient cohorts to facilitate integration into cytologic workflow.

Incomplete surgical excision of keratinocyte skin cancers: a systematic review and meta-analysis.

BACKGROUND: Keratinocyte or nonmelanoma skin cancer (NMSC) is the commonest malignancy worldwide. The usual treatment is surgical excision. Current guidelines underestimate incomplete excision rates. OBJECTIVES: We aimed to determine the risk of incomplete excision of NMSCs through a systematic review and meta-analysis of primary clinical studies. METHODS: A PRISMA-compliant systematic review and meta-analysis was performed using methodology proposed by Cochrane (PROSPERO CRD42019157936). A comprehensive search strategy was applied to MEDLINE, Embase, Scopus, CINAHL, EMCare, Cochrane Library and the grey literature (January 2000-27 November 2019). All studies were included except those on Mohs micrographic surgery, frozen section or biopsies. Abstract screening and data extraction were performed in duplicate. Risk of bias was assessed using a tool for prevalence/incidence studies. The primary outcome was the proportion of incomplete surgical excisions. A random-effects model for pooling of binomial data was used. Differences between proportions were assessed by subgroup meta-analysis and meta-regression, which were presented as risk ratios (RRs). RESULTS: Searching identified 3477 records, with 110 studies included, comprising 53 796 patients with 106 832 basal cell carcinomas (BCCs) and 21 569 squamous cell carcinomas (SCCs). The proportion of incomplete excisions for BCC was 11·0% [95% confidence interval (CI) 9·7-12·4] and for SCC 9·4% (95% CI 7·6-11·4). Proportions of incomplete excisions by specialty were: dermatology, BCCs 6·2% and SCCs 4·7%; plastic surgery, BCCs 9·4% and SCCs 8·2%; general practitioners, BCCs 20·4% and SCCs 18·9%. The risk of incomplete excision for general practitioners was four times that of dermatologists for both BCCs (RR 3·9, 95% CI 2·0-7·3) and SCCs (RR 4·8, 95% CI 1·0-22·8). Studies were heterogeneous (I2  = 98%) and at high risk of bias. CONCLUSIONS: The proportion of incomplete excisions is higher than previously reported. Excisions performed by specialists may lower the risk of incomplete excision.

Educational Case: Aortic Valve Stenosis.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.

Lower mitochondrial DNA content but not increased mutagenesis associates with decreased base excision repair activity in brains of AD subjects.

Accumulation of oxidative mitochondrial DNA (mtDNA) damage and impaired base excision repair (BER) in brains have been associated with Alzheimer's disease (AD). However, it is still not clear how these affect mtDNA stability, as reported levels of mtDNA mutations in AD are conflicting. Thus, we investigated whether alterations in BER correlate with mtDNA instability in AD using postmortem brain samples from cognitively normal AD subjects and individuals who show neuropathological features of AD, but remained cognitively normal (high-pathology control). To date, no data on DNA repair and mtDNA stability are available for these individuals. BER activities, mtDNA mutations, and mtDNA copy number were measured in the nuclear and mitochondrial extracts. Significantly lower uracil DNA glycosylase activity was detected in nuclear and mitochondrial extracts from AD subjects, while apurinic/apyrimidinic endonuclease activity was similar in all groups. Although mtDNA mutation frequency was similar in all groups, mtDNA copy number was significantly decreased in the temporal cortex of AD brains but not of high-pathology control subjects. Our results show that lower mitochondrial uracil DNA glycosylase activity does not result in increased mutagenesis, but rather in depletion of mtDNA in early-affected brain regions during AD development.

Practice Guidelines for Standards of Adult Sleep Medicine Services.

Sleep disorders, i.e. diseases that affect, disrupt or involve sleep, represent major challenges for physicians and healthcare systems worldwide. The high prevalence, the complexity and the health burden of sleep disorders demand the establishment of specific clinical sleep centres where adequate and efficient diagnosis and management of patients with such diseases can be provided. This document describes practice guidelines for standards of adult sleep medicine centres in Ireland. These guidelines are the result of a consensus procedure in which all committee members of the Irish Sleep Society (ISS) were involved. The scope of these guidelines is to define the requirements of sleep medicine services, in terms of personnel, facilities, equipment and procedures.

Multiple Molecular Mechanisms Rescue mtDNA Disease in C. elegans.

Genetic instability of the mitochondrial genome (mtDNA) plays an important role in human aging and disease. Thus far, it has proven difficult to develop successful treatment strategies for diseases that are caused by mtDNA instability. To address this issue, we developed a model of mtDNA disease in the nematode C. elegans, an animal model that can rapidly be screened for genes and biological pathways that reduce mitochondrial pathology. These worms recapitulate all the major hallmarks of mtDNA disease in humans, including increased mtDNA instability, loss of respiration, reduced neuromuscular function, and a shortened lifespan. We found that these phenotypes could be rescued by intervening in numerous biological pathways, including IGF-1/insulin signaling, mitophagy, and the mitochondrial unfolded protein response, suggesting that it may be possible to ameliorate mtDNA disease through multiple molecular mechanisms.

Age-related accumulation of phosphorylated mitofusin 2 protein in retinal ganglion cells correlates with glaucoma progression.

Dysregulation of axonal bioenergetics is likely a key mechanism in the initiation and progression of age-related neurodegenerative diseases. Glaucoma is a quintessential neurodegenerative disorder characterized by progressive deterioration of the optic nerve (ON) and eventual death of retinal ganglion cells (RGCs). Age and elevation of intraocular pressure are key risk factors in glaucoma, but the common early hallmarks of decreased axonal transport and increased bioenergetic vulnerability likely underlie disease initiation. We examined the correlation between bioenergetics and axonal transport with mitochondrial mutation frequency and post-translational modifications of mitofusin 2 (Mfn2) in RGCs during glaucoma progression. No increase in the frequency of mtDNA mutations was detected, but we observed significant shifts in mitochondrial protein species. Mfn2 is a fusion protein that functions in mitochondrial biogenesis, maintenance, and mitochondrial transport. We demonstrate that Mfn2 accumulates selectively in RGCs during glaucomatous degeneration, that two novel states of Mfn2 exist in retina and ON, and identify a phosphorylated form that selectively accumulates in RGCs, but is absent in ON. Phosphorylation of Mfn2 is correlated with higher ubiquitination, and failure of the protein to reach the ON. Together, these data suggest that post-translational modification of Mfn2 is associated with its dysregulation during a window of metabolic vulnerability that precedes glaucomatous degeneration. Future work to either manipulate expression of Mfn2 or to prevent its degradation could have therapeutic value in the treatment of neurodegenerative diseases where long-tract axons are vulnerable.

Cyclophosphamide leads to persistent deficits in physical performance and in vivo mitochondria function in a mouse model of chemotherapy late effects.

Fatigue is the symptom most commonly reported by long-term cancer survivors and is increasingly recognized as related to skeletal muscle dysfunction. Traditional chemotherapeutic agents can cause acute toxicities including cardiac and skeletal myopathies. To investigate the mechanism by which chemotherapy may lead to persistent skeletal muscle dysfunction, mature adult mice were injected with a single cyclophosphamide dose and evaluated for 6 weeks. We found that exposed mice developed a persistent decrease in treadmill running time compared to baseline (25.7±10.6 vs. 49.0±16.8 min, P = 0.0012). Further, 6 weeks after drug exposure, in vivo parameters of mitochondrial function remained below baseline including maximum ATP production (482.1 ± 48.6 vs. 696.2 ± 76.6, P = 0.029) and phosphocreatine to ATP ratio (3.243 ± 0.1 vs. 3.878 ± 0.1, P = 0.004). Immunoblotting of homogenized muscles from treated animals demonstrated a transient increase in HNE adducts 1 week after exposure that resolved by 6 weeks. However, there was no evidence of an oxidative stress response as measured by quantitation of SOD1, SOD2, and catalase protein levels. Examination of mtDNA demonstrated that the mutation frequency remained comparable between control and treated groups. Interestingly, there was evidence of a transient increase in NF-ĸB p65 protein 1 day after drug exposure as compared to saline controls (0.091±0.017 vs. 0.053±0.022, P = 0.033). These data suggest that continued impairment in muscle and mitochondria function in cyclophosphamide-treated animals is not linked to persistent oxidative stress and that alternative mechanisms need to be considered.

Mass cytometry identifies a distinct monocyte cytokine signature shared by clinically heterogeneous pediatric SLE patients.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease with heightened disease severity in children. The incomplete understanding of the precise cellular and molecular events that drive disease activity pose a significant hurdle to the development of targeted therapeutic agents. Here, we performed single-cell phenotypic and functional characterization of pediatric SLE patients and healthy controls blood via mass cytometry. We identified a distinct CD14hi monocyte cytokine signature, with increased levels of monocyte chemoattractant protein-1 (MCP1), macrophage inflammatory protein-1ß (Mip1ß), and interleukin-1 receptor antagonist (IL-1RA). This signature was shared by every clinically heterogeneous patient, and reproduced in healthy donors' blood upon ex-vivo exposure to plasma from clinically active patients only. This SLE-plasma induced signature was abrogated by JAK1/JAK2 selective inhibition. This study demonstrates the utility of mass cytometry to evaluate immune dysregulation in pediatric autoimmunity, by identification of a multi-parametric immune signature that can be further dissected to delineate the events that drive disease pathogenesis.

The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2-rearranged BCP-ALL.

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Massively parallel digital transcriptional profiling of single cells.

Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3' mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.

Regulation of IL-12p40 by HIF controls Th1/Th17 responses to prevent mucosal inflammation.

Intestinal inflammatory lesions are inherently hypoxic, due to increased metabolic demands created by cellular infiltration and proliferation, and reduced oxygen supply due to vascular damage. Hypoxia stabilizes the transcription factor hypoxia-inducible factor-1α (HIF) leading to a coordinated induction of endogenously protective pathways. We identified IL12B as a HIF-regulated gene and aimed to define how the HIF-IL-12p40 axis influenced intestinal inflammation. Intestinal lamina propria lymphocytes (LPL) were characterized in wild-type and IL-12p40-/- murine colitis treated with vehicle or HIF-stabilizing prolyl-hydroxylase inhibitors (PHDi). IL12B promoter analysis was performed to examine hypoxia-responsive elements. Immunoblot analysis of murine and human LPL supernatants was performed to characterize the HIF/IL-12p40 signaling axis. We observed selective induction of IL-12p40 following PHDi-treatment, concurrent with suppression of Th1 and Th17 responses in murine colitis models. In the absence of IL-12p40, PHDi-treatment was ineffective. Analysis of the IL12B promoter identified canonical HIF-binding sites. HIF stabilization in LPLs resulted in production of IL-12p40 homodimer which was protective against colitis. The selective induction of IL-12p40 by HIF-1α leads to a suppression of mucosal Th1 and Th17 responses. This HIF-IL12p40 axis may represent an endogenously protective mechanism to limit the progression of chronic inflammation, shifting from pro-inflammatory IL-12p70 to an antagonistic IL-12p40 homodimer.

An anatomical study of the intersigmoid fossa and applications for internal hernia surgery.

PURPOSE: To improve the knowledge of the morphometry and the surrounding anatomical structures of the intersigmoid fossa and to determine possible surgical applications. METHOD: Forty eight adult cadavers (29 female and 19 male; mean age 83 years) underwent dissection in the Laboratoire d'Anatomie des Alpes Francaises. Two injections in the right carotid resulted in a total body concentration of formalin of 1.3 %. The study parameters were the dimensions of the intersigmoid fossa orifice and the fossa's relationship to surrounding structures. Data were recorded and analyzed using Microsoft Office Excel (MS Cerp). A Pearson coefficient r was used to examine the correlation between the length of colon and the ISF volume. RESULTS: The intersigmoid fossa was present in 75 % of cases (n = 36). The average dimensions for the transverse diameter, longitudinal diameter, and the depth were, respectively, 20.5 ± 0.2, 20.3 ± 0.13, and 26.8 ± 0.2 mm. The primary and secondary roots bordering this fossa measured on average 59.1 ± 0.1 and 48.3 ± 0.13 mm. In 13.9 % of cases (n = 5), the maximum depth was >40 mm and in 16.7 % of cases (n = 6), one of the diameters of the orifice entry of the fossa was >40 mm. The ureter and external iliac artery were the most frequently encountered structures during the dissection of the fundus of the intersigmoid fossa. CONCLUSION: The intersigmoid fossa remains present in most of the reported dissections of cadavers. It constitutes an essential landmark in the surgery of the sigmoid colon due to its deep structural relationship with the left ureter and external iliac artery.