Lung cancer is the major cause of cancer related deaths in the world. New therapies have improved outcomes. Unfortunately, overall 5 year survival is ~20%. Therefore, better understanding of tumor biology and the microenvironment may lead to new therapeutic targets. The lung microbiome has recently emerged as a major mediator of host inflammation and pathogenesis. Understanding how the lung microbiota exerts its effects on lung cancer and the tumor microenvironment will allow for novel development of therapies.Lung cancer is the leading cause of cancer-related death. Over the past 5–10 years lung cancer outcomes have significantly improved in part due to better treatment options including immunotherapy and molecularly targeted agents. Unfortunately, the majority of lung cancer patients do not enjoy durable responses to these new treatments. Seminal research demonstrated the importance of the gut microbiome in dictating responses to immunotherapy in melanoma patients. However, little is known regarding how other sites of microbiota in the human body affect tumorigenesis and treatment responses. The lungs were traditionally thought to be a sterile environment; however, recent research demonstrated that the lung contains its own dynamic microbiota that can influence disease and pathophysiology. Few studies have explored the role of the lung microbiome in lung cancer biology. In this review article, we discuss the links between the lung microbiota and cancer, with particular focus on immune responses, metabolism and strategies to target the lung microbiome for cancer prevention. Lung cancer is the leading cause of cancer-related death in the world. Even with the advancement in recent therapeutics and the development of immunotherapy, the 5-year overall survival in patients with non-small lung cancer (NSCLC) is <20%. Nonetheless, recent excitement for immunotherapy in lung cancer has centered around a minority of patients who develop durable, long-lasting responses to treatment, even in patients with advanced stage disease.Moreover, a recent study demonstrated the importance of the gut microbiome in predicting response to immunotherapy in melanoma patients . The lunTherefore, in this review we will summarize the current research surrounding investigation of the lung microbiome in lung diseases with a focus on lung cancer. Additionally, we will aim to elucidate the potential relationship between the lung microbiome, inflammation and metabolism. Finally, we will discuss potential therapeutic interventions that may arise out of further research investigating the role of the lung microbiome in lung cancer patients with a focus on how the lung microbiome may be exploited to improve responses and outcomes to immunotherapy.4–106 bacterial cells per cubic meter [For many decades it was believed the lung represented a sterile environment. With the advent of culture-independent techniques and high-throughput sequencing of the 16S rRNA gene, a small highly conserved cistron of the bacterial genome, it became possible to identify the presence of bacterial communities at the genus or species-level phylogeny. In a landmark study in 2010, the authors published the first application of culture-independent techniques to identify microbiota present within the lungs of healthy patients as well as patients with asthma and chronic obstructive pulmonary disease (COPD) . To dateic meter . It is wic meter ,8. Elimiic meter ,10,11,12ic meter . The numPrevotella, Veillonella and Streptococcus [In general, studies indicate that a relatively uniform microbiota composes the healthy lung with the presence of certain dominant taxonomic groups . The mostococcus ,14,15. Itococcus ,17. WithPrevious studies have linked the microbiota to various malignancies. More recently, studies performed by the Wargo group have correlated gut microbial composition to response to chemotherapy and immunotherapy in colon cancer models and advanced stage melanoma patients ,18. TherMycobacterium tuberculosis (TB) to lung cancer [Haemophilus influenza, Enterobacter and Escherichia coli [n = 8) as well as female, never smokers without cancer (n = 8). Samples taken from the lung cancer cohort had enrichment of Granulicatella, Abiotrophia and Streptococcus genera compared to the non-cancer cohort [Veillonella, Neisseria, Capnocytophaga and Selenomonas in sputum collected from squamous cell and adenocarcinoma lung cancer patients (n = 20) as compared to non-cancer control subjects (n = 10) [n = 4) and non-lung cancer subjects (n = 6) and found that seven specific bacterial species were present in all samples with significantly higher levels of enrichment of Streptococcus viridans in lung cancer samples [Veillonella and Megasphaera, in lung cancer patients as well as Firmicutes and TM7 phyla as compared to patients with benign pathology [Streptococcus and Veillonella) as compared to non-cancer and healthy subjects. Interestingly, the uninvolved cancer airway samples shared many similar findings as seen in the involved cancer airway samples. It should be noted that this study included rigorous “control” specimens including buccal samples and bronchoscopy scope washes prior to procedures [Characterization of the lung microbiome and its influences on lung cancer and treatment is in its infancy. Epidemiological studies have correlated g cancer ,27,28. Ag cancer ,30. Othehia coli . It shour cohort . In addi(n = 10) . In a pi samples . More reathology . Additioocedures . Togethen = 24) and healthy subjects (n = 18). Tissue from lung cancer patients had a significant decrease in microbial diversity as compared to healthy controls. Streptococcus and Staphylococcus were significantly more abundant in cancer patients compared to controls. The authors also found that the abundance of Staphylococcus and Dialister were highest in healthy subjects and declined in noncancerous tissues of lung cancer patients reaching lowest levels in tumor tissues [Sphingomonas, Psychromonas and Serratia while the abundance of Cloacibacterium, Geobacillus and Brevibacterium were reduced [Thermus genus while Legionella was higher in patients who developed metastatic disease [Further analysis has involved understanding differences between microbial presence in cancerous lung versus noncancerous tissue. In one study, paired samples from tumor tissue and contralateral noncancerous sites were collected from lung cancer patients  and resident dendritic cells (DC) as well as other immune cells that monitor the lower airways for pathogenic insults. They are critical mediators of lung immune homeostasis ensuring that inflammatory and immune responses are activated in response to a pathogenic insult while dampening responses to harmless environmental stimuli. In general, the lung microenvironment is one of high immune tolerance. Both AMs and DCs stimulate the proliferation of regulatory T cells (Treg) and release prostaglandin E2 (PGE2), tumor growth factor-beta (TGF-B) and interleukin-10 (IL-10) which leads to a tolerogenic state ,52. FurtOn the other hand, pathogenic bacteria with virulence factors can easily breach the mucous layer and infiltrate the epithelium leading to an inflammatory response . TogetheStaphylococcus aureus, an upper respiratory tract (URT) colonizer, and found that it was essential for augmenting resistance to lethal inflammatory responses to influenza viral challenges. SPF mice were found to have less S. aureus as compared to mice living in the natural environment and subsequently succumbed to death at higher rates due to induction of acute inflammation after influenza viral challenges. Furthermore, it was shown that S. aureus recruits monocytes into alveoli and induces polarization of AMs to an M2 phenotype leading to suppression of lethal inflammatory responses through release of anti-inflammatory molecules in response to influenza insult [Prevotella and Veillonella (upper respiratory tract (URT) colonizers) termed supraglottic predominant taxa (SPT) and another population of persons termed background predominant taxa (BPT) characterized by low bacterial copy number whose BALF resembled background taxa environmental microbiota. Subjects with pneumotype SPT demonstrated increased numbers of lymphocytes in BALF, increased Th17 cells, IL-1α, IL-1β, fractalkine, IL-17, free fatty acids, inflammatory pathway mRNA and blunted TLR4 response. There was also decreased β-diversity and increased bacterial abundance [Prevotella and Veillonella) in regulating the level of airway inflammation and Th17 immune activation in the lower airways. Additionally, co-culture experiments in which Prevotella and Haemophilus species were cultured with human DCs led to the observation that Prevotella could suppress Haemophilus induction of IL-12p70 in DCs leading to modulation of the immune response [Prevotella-dominant airway microbiota was associated with development of more inflammatory prone macrophages [Prevotella-predominant microbiota in the lower airways could generate an immune microenvironment enriched with Th17 cell populations and IL-17 production which could support tumorigenesis. Indeed, IL-17A T cells have been linked to tumor invasion and metastasis in lung adenocarcinoma [As suggested, the lung microbiota is thought to play a role in immune tolerance by influencing APCs and Treg recruitment. Gollwitzer et al. found that a progressive shift from Gammaproteobacteria and Firmcutes towards Bacteroidetes in neonates induces increased expression of programmed-death ligand 1 (PD-L1) on dendritic cells which lead to necessary Treg development after birth . Anothera insult . This sta insult . The autbundance . Interesbundance  again suresponse . Anotherrophages . This suarcinoma .Herbaspirillum and Sphingomonadaceae not observed in the lungs of non-tumor bearing mice [Streptococcus and enrichment of Proteobacteria and Actinobacteria [Lung cancer is closely associated with chronic inflammation and the immune microenvironment is characterized by the accumulation of pro-inflammatory cytokines and pro-tumorigenic factors that can aid in tumorigenesis . As discing mice . These fing mice . Togethebacteria . These sPrevotella, Veillonella and Streptococcus [Veillonella was cultured induced expression of ERK, PTEN, VEGFA and certain genes important to inflammasome function in A549 lung adenocarcinoma cells grown with the cultured media, suggesting that bacterial byproducts may be important for modulation of tumorigenic transcriptional pathways. While there are limited studies on lung microbiota-derived byproducts and the effects on host immune response as well as tumorigenesis, there have been a number of studies examining this relationship with regards to the gut microbiota. For example, bacterial derived acetaldehyde is known to be a carcinogen and deoxycholic acid (DCA) produced from the gut microbiota of obese individuals has been associated with hepatocellular carcinoma development [Clostridium species present in the gut have been linked to the production of SCFAs and development of Treg cells [One way the lung microbiota may exert it effects on immune cells and tumor are through microbial byproducts. Interestingly, Tsay et al. demonstrated that lung cancer patients had enrichment of the upper respiratory tract flora, especially tococcus . When thelopment ,83. Moreelopment . Clostrieg cells ,86,87. Oeg cells ,89. TherEscherichia coli and S. aureus may be able to increase proteases and protease inhibitors important to infection and immune response [Prevotella, Veillonella and Streptococcus had a relative increase in abundance of carbohydrate metabolism genes and decreased cellobiose and fucose-rhamnose indicative of active bacterial metabolism [Additionally, the gut microbiome has been demonstrated to have direct effects on host metabolism which represents another way the microbiota may influence the local microenvironment . Gut micresponse . Furtherresponse . Recentlresponse . Bacteritabolism . In additabolism . Therefotabolism . TogetheUnderstanding the interplay between the lung microbiota and immune and tumor microenvironment will allow for potential new therapeutic strategies. These therapies could be designed to modulate commensal lung microbiota to induce a more tumor suppressive environment, target specific bacterial enzymes or byproducts important for either tumor growth, onco-immune responses, or both, modulate the resultant host biological effects induced by the lung microbiota or targeting the mechanism by which bacteria immigrate to the lung.Lactobacilli was found to stimulate respiratory immunity and increase resistance towards viral infections [Streptococcus salivarius has been shown to prevent acute otitis [As discussed altered lung microbiota have the ability to attenuate immune responses in immune cells with certain species linked to the polarization of suppressive alveolar macrophages and decrease response to lipopolysaccharide leading to resistance to acute inflammation while other species have been linked to increased Th17 cell-mediated lung inflammation and decreased inflammation ,71,96. Tfections ,103. In e otitis ,104. In e otitis . This suPseudomonas aeruginosa otitis media or externa [Achromobacter infection successfully while a small study compared administration of aerosolized BPs versus conventional antibiotic treatment for cystic fibrosis patients with Pseudomonas aeruginosa infection and found BPs reduced Pseudomonas aeruginosa concentration as well as need for additional antibiotics [In addition, bacteriophages (BPs) could potentially be engineered against specific lung microbiota in order to shift the tumor and immune microenvironment in lung cancer patients towards a more anti-tumorigenic environment. BPs are viruses that can infect and kill bacteria but do not infect human or host cells. BPs are able to be genetically engineered to target specific bacterial species ,106. Num externa . Aerosolibiotics ,111. To 0F1 ATP synthase [Targeting specific bacterial enzymes may also provide a way to modulate the lung microbiota and its effects on the tumor microenvironment. Bacteria, not unlike eukaryotic cells, rely on the biosynthesis of proteins, RNA, DNA as well as metabolites for their survival. In fact, microbial pathogenicity requires metabolic pathways in order to support bacterial growth and bacterial pathogens reprogram their metabolism to allow for survival . Traditisynthase . While csynthase . By undePrevotella, Veillonella and Streptococcus) leads to a Th17/neutrophilic phenotype within the lung microenvironment. Therefore, it can be surmised that lung microbiota enriched with URT microbes will support a pro-tumorigenic microenvironment through induction of Th17 responses. In the future, targeted treatments towards IL-17 combined with immunotherapy may yield increased responses to therapy in lung cancer patients. Indeed, Jin et al. found significantly decreased tumor growth, IL-1β and neutrophil infiltration when KP mice were treated with an IL-17A neutralizing antibody [Streptococcus and Veillonella genera are classified under the Firmicutes phylum and again this suggests that presence of URT microbes in the lower airway is associated with a pro-inflammatory phenotype. Furthermore, IL-6 is known to play an essential role in lung cancer by promoting COPD-like inflammation [As described, lung cancer tumorigenesis is characterized by an immune microenvironment enriched by Th17 cell responses along with expression of IL-17 as well as other cytokines. Additionally, lung commensal microbiota enriched with upper respiratory tract microbes  to investigate the presence of unique microbial signatures in tissue and blood within and between 33 types of cancer from treatment-naïve patients. The authors were able to determine that blood-based microbial DNA signatures could discriminate within and between most types of cancer, including low-grade tumors, using commercial-grade cell-free tumor DNA platforms . TogetheIt is well accepted that the lung harbors a dynamic microbiota that is influenced by a number of host and environmental factors. With the recent studies demonstrating the importance of the gut microbiota in predicting responses to immunotherapy in melanoma patients there has been renewed interest in understanding how the microbiome, including the lung commensal microbiota, modulates and remodels the tumor microenvironment. Further studies elucidating the role of the lung microbiome in metabolic and inflammatory regulation within the lungs of lung cancer patients may lead to the development of medical interventions designed to improve outcomes of treatments such as immunotherapy and aid in the evolution of preventative strategies for lung cancer patients.


Logopenic progressive aphasia is a neurodegenerative syndrome characterized by sentence repetition and naming difficulties arising from left-lateralized temporoparietal atrophy. Clinical descriptions of logopenic progressive aphasia largely concentrate on profiling language deficits, however, accumulating evidence points to the presence of cognitive deficits even on tasks with minimal language demands. Although non-linguistic cognitive deficits in logopenic progressive aphasia are thought to scale with disease severity, patients at discrete stages of language dysfunction display overlapping cognitive profiles, suggesting individual-level variation in cognitive performance, independent of primary language dysfunction. To address this issue, we used principal component analysis to decompose the individual-level variation in cognitive performance in 43 well-characterized logopenic progressive aphasia patients who underwent multi-domain neuropsychological assessments and structural neuroimaging. The principal component analysis solution revealed the presence of two, statistically independent factors, providing stable and clinically intuitive explanations for the majority of variance in cognitive performance in the syndrome. Factor 1 reflected ‘speech production and verbal memory’ deficits which typify logopenic progressive aphasia. Systematic variations were also confirmed on a second, orthogonal factor mainly comprising visuospatial and executive processes. Adopting a case-comparison approach, we further demonstrate that pairs of patients with comparable Factor 1 scores, regardless of their severity, diverge considerably on visuo-executive test performance, underscoring the inter-individual variability in cognitive profiles in comparably ‘logopenic’ patients. Whole-brain voxel-based morphometry analyses revealed that speech production and verbal memory factor scores correlated with left middle frontal gyrus, while visuospatial and executive factor scores were associated with grey matter intensity of right-lateralized temporoparietal, middle frontal regions and their underlying white matter connectivity. Importantly, logopenic progressive aphasia patients with poorer visuospatial and executive factor scores demonstrated greater right-lateralized temporoparietal and frontal atrophy. Our findings demonstrate the inherent variation in cognitive performance at an individual- and group-level in logopenic progressive aphasia, suggesting the presence of a genuine co-occurring cognitive impairment that is statistically independent of language function and disease severity. et al. found systematic inter-individual variation in language and non-linguistic general cognitive performance in patients with logopenic progressive aphasia, a patient group conceptualized as showing predominant naming and repetition deficits. These impairments were independent of disease severity and degree of aphasia and were associated with distinct, non-overlapping neural correlates.Ramanan   Logopenic progressive aphasia (LPA) is a rare neurodegenerative brain disorder, the canonical features of which centre on language dysfunction, including slowing in spontaneous speech, phonological errors and paraphasias, sentence repetition, sentence comprehension and word-finding difficulties . Our primary aims were to reveal the extent of graded variations in cognitive performance within the LPA syndrome, and to use the emergent components to characterize patient performance at the individual level. We predicted that marked cognitive heterogeneity would be evident, regardless of the severity of language impairments. Finally, we sought to establish the neural substrates of the graded variation in cognitive performance within the LPA syndrome, using voxel-based morphometry (VBM).To this end, we employed PCA to explore the neurocognitive architecture of language and general cognitive performance in a large well-characterized sample of LPA patients  completed the ACE-III  with false discovery rate correction for multiple comparisons , Chi-squared tests were used. For all continuous variables , normality of distribution was examined using the Shapiro–Wilk tests and box-and-whisker plots. Accordingly, parisons  were useAll subsequent statistical analyses were conducted in the LPA group. As PCA algorithms operate on standardized datasets with no missing variables, the frequency of missing neuropsychological data was first tabulated and plotted for subsequent imputation . Across k-fold cross-validation approach  PCA. Varimax rotation facilitates interpretations of PCA output by maximizing the dispersion of factor loadings between components, allowing for a little variance to be shared commonly between emergent components. In line with standard approaches , factorsIt must be noted that factor names are simply shorthand labels that reflect the majority of cognitive tests loading onto that particular factor, and by no means reflect the entirety of cognitive processes that underpin performance on each test loading onto that particular factor. Individual patient scores on each factor were extracted and used as orthogonal covariates in subsequent neuroimaging analyses. In addition, we projected the lower bound of normality (i.e. −1.96 standard error of the mean) from the control data into the patients’ PCA space to facilitate behavioural interpretation of patient factor scores relative to control test performance . By contrast, tests that loaded heavily on the orthogonal factor (Factor 2) reflected measures on which performance is traditionally thought to be affected in later stages of LPA . We therefore treated each patient’s Factor 1 score as a simple metric of how ‘logopenic’ they are and used these scores to predict test performance on neuropsychological measures loading differentially on Factors 1 and 2. This comparison would demonstrate how comparably logopenic patients (with similar Factor 1 scores) diverge on test measures posited to be relatively preserved, until later stages of LPA.x-axis . All 3D T1-weighted images were acquired using the following sequences: coronal acquisition, matrix 256 × 256 mm, 200 slices, 1 mm isotropic voxel resolution, echo time/repetition = 2.6/5.8 ms, flip angle α = 8°.Sixty-three participants (35 LPA and 28 Controls) underwent structural Thttps://www.fil.ion.ucl.ac.uk/spm/software/spm12/, accessed 26 August 2020). Full details of the standard pre-processing pipeline are provided in We used combined grey and white matter VBM to account for co-occurring cortical grey and subcortical white matter changes that are prototypical of neurodegenerative disease syndromes such as LPA .Voxel-wise differences of grey and white matter intensity between LPA and Control groups were assessed using independent VBM correlation analyses are entirely constrained by variations in voxel-level intensity and test performance. In the context of progressive diseases, this means that highly atrophic regions that subsequently have uniformly low voxel-level variance are unlikely to emerge in the correlation analyses as they are consistently affected across cases. These regions, nevertheless, could be critical to explaining the observed behavioural profile and therefore, it is important to interpret VBM results in the context of whole-brain voxel-level variance. To complement our atrophy analyses, we therefore computed voxel-level inter-subject variance maps of grey and white matter intensity for all participants. The resultant whole-brain images were further masked to consider only clusters emerging in our atrophy analyses. As before, age and total intracranial volume were regressed out as nuisance variables prior to extracting variance maps.P-values > 0.1). When compared to patients with higher Factor 1 scores, those with lower Factor 1 scores had greater disease severity , whilst the difference of disease duration was not statistically significant . In contrast, no significant group differences were noted on disease severity  and disease duration  between patients split on Factor 2 scores. Regression models with separate directional contrasts (i.e. independent t-tests) were used to assess differences in cortical grey matter and subcortical white matter intensities between LPA subgroups (i.e. high and low scorers) on each Factor score, with age and total intracranial volume included as nuisance variables. Clusters were extracted at P < 0.001, uncorrected, with a cluster threshold of 100 contiguous voxels.We further investigated whole-brain changes in grey and white matter intensity in patients with ‘low’ and ‘high’ factor scores. Patients were stratified into two folds on either end of a zero score on Factor 1 and Factor 2 each see . StratifN = 35) were employed to examine associations between whole-brain grey and white matter intensity and PCA-generated factor scores. A correlation-only statistical model was implemented for additional statistical power, using t-contrasts to measure associations between grey and white matter intensity and PCA-generated factor scores. Age and total intracranial volume were included as nuisance covariates in the analyses. Anatomical locations of statistical significance were overlaid on the Montreal Neurological Institute (MNI) standard brain with maximum co-ordinates provided in MNI stereotaxic space. Clusters were extracted using a threshold of P < 0.001 uncorrected for multiple comparisons with a cluster threshold of 100 contiguous voxels.Finally, correlation analyses within the LPA group (https://osf.io/bn534/). No part of the study procedures or analyses were preregistered prior to the research being undertaken.The ethical requirement to ensure patient confidentiality precludes public archiving of our data. Researchers who would like to access the raw data should contact the corresponding authors, who will liaise with the ethics committee that approved the study, and accordingly, as much data that are required to reproduce the results will be released to the individual researcher. The code used for this project has been made available for review on the Open Science Framework website . LPA patients performed significantly worse than controls on measures of global cognition, as well as targeted neuropsychological assessments of episodic memory, semantic naming and comprehension, single-word repetition, visuo-constructional abilities and executive function . These profiles are in keeping with previous descriptions of the LPA cognitive profile . No other significant correlations emerged between neuropsychological test performance and disease severity in LPA , phonological working memory , naming  and repetition (SYDBAT Repetition and Digit Span Forward and Backward) . TogetheOur PCA analyses further suggested the presence of an orthogonal set of variations on a second factor. Factor 2 mainly loaded on measures of executive (Trails Time Difference), attention  and visuospatial  abilities. In addition, the SYDBAT Comprehension subtest performance also loaded onto this factor. For brevity, we refer to this factor as the ‘visuospatial and executive factor’. Importantly, patients with both high and low Factor 1 scores exhibited uniform variation on Factor 2 scores and this variation was noted both proximally and distally from the lower bound of normal control performance . TogetheIn summary, our PCA pointed to the existence of two orthogonal sets of variations in neuropsychological performance in LPA. While the first factor resembles the classic language profile of LPA, the uniform distribution of scores on Factor 2 suggests a co-occurring primary disruption of visuospatial and executive processes in this syndrome.r = −0.25; P = 0.1) or visuospatial and executive factor  , but not with the visuospatial and executive factor  (No significant correlations were found between disease severity (CDR-FTLD SoB) and scores on the speech production and verbal memory factor  . In contP > 0.1) . The lacIn a second step, we aimed to demonstrate how patients who present as ‘comparably logopenic’ can show divergent visuospatial and executive performance. For this, we first chose LPA patient pairs with comparable Factor 1 scores , predicted and actual scores were nearly similar across all patient pairs (except for pair 4 on SYDBAT Naming) . This paGroup differences in grey and white matter intensity are presented in Visual inspection of variance maps revealed that variance in whole-brain grey and white matter intensity was lowest in left perisylvian regions, typically affected in the earliest stages of LPA . ExaminiGroup differences in grey and white matter intensity are presented in Associations between grey and white matter intensity and factor scores in the LPA group are displayed in In the overall LPA group, speech production and verbal memory factor scores were found to correlate with grey matter intensity of the left middle frontal gyrus  and medial parietal , right lateral temporal regions  and the right middle frontal gyrus. Additionally, a small cluster in the ventral temporal cortex  extending into the right cerebellar cortex was noted. Changes in white matter intensity of the right superior longitudinal fasciculus  and right middle/inferior longitudinal fasciculus  were further found to correlate with visuospatial and executive factor scores  correlating with grey matter intensity of the middle frontal gyrus, and the visuospatial and executive factor scores (Factor 2) correlating with largely right-sided temporoparietal and frontal regions and their underlying white matter connections. Importantly, the regions to emerge as significant in our covariate analyses  are not This study demonstrates that the presence of visuospatial and executive deficits in LPA, beyond core language disturbance, does not reflect advancing disease severity. Instead, these deficits in LPA form their own independent cognitive dimension with discrete neuroanatomical bases and are reliably present even in the early stages of LPA. In more detail, the PCA identified two emergent factors capturing the heterogeneity of the LPA cognitive profile. The first factor reflected the expressive language and phonological working memory impairments that are not only diagnostic of LPA  diverge considerably on their visuo-executive performance. Importantly, this pattern was present even when comparing pairs of LPA patients with mild, moderate or severe language difficulties, suggesting attention, executive and visuospatial deficits are core features of the LPA syndrome. From a clinical standpoint, our findings align well with previous descriptions of single cases of LPA presenting with ‘atypical’ symptoms. For example, single cases of LPA have been described to present with a marked breakdown in attentional processing manifesting in hemi-spatial neglect . SimilarWe next explored associations between factor scores and cortical and subcortical brain changes in LPA. Performance on the speech production and verbal memory factor was found to correlate with grey and white matter changes of the left middle frontal gyrus. This region is a key frontal node of the language and executive processing networks, with well-described roles in supporting fluency in expressive language . Disruptin vivo confirmation of underlying Alzheimer’s pathology revealed marked single-word comprehension difficulties in LPA (Turning our attention to Factor 2, performance on the visuospatial and executive factor was found to correlate with grey and white matter intensity of right-lateralized temporoparietal and prefrontal regions, including precentral, inferior parietal, lateral temporal, inferior frontal and insular cortices. Moreover, LPA patients with poorer scores on the visuospatial and executive factor tended to demonstrate greater right-hemisphere temporoparietal and prefrontal involvement. Right-lateralized regions such as precentral gyrus and superior/inferior parietal regions are typically proposed to regulate goal-directed and stimulus-driven attentional abilities , while mP < 0.001, however, this threshold is far more conservative than traditional multiple comparison approaches such as false discovery rate and is increasingly used when exploring links between cognition and neurodegeneration (The current findings must be interpreted in the context of certain caveats. First, the majority of our LPA patients have not yet come to autopsy, precluding confirmation of underlying Alzheimer versus non-Alzheimer pathology in our cohort. Nevertheless, we rigorously applied the diagnostic criteria of LPA (Despite these limitations, our findings hold important clinical implications relevant to the diagnosis and characterization of LPA. Identification of heterogeneity in cognitive function in LPA underscores the need for comprehensive neuropsychological workup beyond language in primary progressive aphasia. By limiting their primary focus to language impairments, clinicians will underestimate the presence and severity of visuospatial and executive impairments in LPA, potentially leading to increased functional disturbances and carer burden. We further speculate that the emergence of visuospatial and executive impairments in LPA can be thought of as converse to atypical variants of Alzheimer’s disease such as posterior cortical atrophy. Although described as a syndrome with preponderant visual disturbances due to early right-sided parietal atrophy, posterior cortical atrophy patients gradually demonstrate increasing language and verbal working memory dysfunction (In conclusion, we provide new insights into the syndrome of LPA, by revealing a fundamental impairment of visuospatial and executive processes, independent of the characteristic language difficulties in this syndrome. This visuospatial and executive impairment varies systematically across LPA patients, irrespective of disease severity and correlates with right-lateralized temporoparietal and frontal regions. Our findings reveal the inherent complexity of the LPA syndrome in terms of cognitive profiles and neural atrophy patterns and suggest that reconceptualization of the LPA syndrome and its relationship to typical and atypical variants of Alzheimer’s disease is warranted.Brain Communications online.fcaa125_Supplementary_DataClick here for additional data file.


Menthol vascular actions in vivo seem to depend on whether the vascular territory under analysis has been directly provoked with menthol or is located deep/distant to the application site. Menthol increases perfusion of directly provoked skin regions due to a complex interplay of increased nitric oxide (NO), endothelium-derived hyperpolarization factors (EDHFs) and sensory nerve responses. In non-provoked vascular beds menthol decreases perfusion which might be attributed to heat-conservation sympathetically-mediated vasoconstriction, although an increase in tissue evaporative heat loss due the formulation ethanol may also play a role. There is increasing evidence that several of menthol’s cellular targets are involved in cardiovascular diseases, such as hypertension. Thus menthol and pharmacologically-similar drugs can play important preventive and therapeutic roles, which merits further investigation.Menthol is a monoterpene alcohol, widely used in several food and healthcare products for its particular odor and flavor. For some decades, menthol has been known to act on the vasculature directly in the endothelium and vascular smooth muscle, with recent studies showing that it also evokes an indirect vascular response via sensory fibers. The mechanisms underlying menthol’s vascular action are complex due to the diversity of cellular targets, to the interplay between signaling pathways and to the variability in terms of response. Menthol can evoke either a perfusion increase or decrease  Peppermint essential oil has been used throughout the centuries for its therapeutic properties in a variety of medical disorders including gastrointestinal, respiratory, reproductive and inflammatory, among others , and is responsible for their distinctive cool/fresh odor and flavor . The maig others . Mentholg others .Menthol (5-methyl-2-propan-2-ylcyclohexan-1-ol) possesses three stereogenic centers and, therefore, four pairs of optical isomers: (+)- and (−)-menthol, (+)- and (−)-neomenthol, (+)- and (−)-neoisomenthol, and (+)- and (−)-isomenthol, whose structures are shown in Menthol absorbed via the gastrointestinal tract, skin or airways is transported to the liver by the circulation, where it is mainly metabolized. When taken orally, menthol undergoes marked rapid absorption in the small intestine , whereasth century with the first publications appearing in the 1920s. For some decades, menthol has been known to act on the vasculature directly in the endothelium or vascular smooth muscle (VSM), with recent studies showing that it also evokes an indirect vascular response via sensory fibers. The mechanisms underlying menthol’s vascular action are complex due to the diversity of cellular targets, to the interplay between signaling pathways and to the variability in terms of response. Menthol can evoke either a perfusion increase or decrease in vivo in different vascular territories, an observation that warrants a critical discussion. The next subsections present a comprehensive review of the main vascular actions attributed to menthol both in vitro and in vivo , along with a critical discussion of the main in vivo findings to clarify these seemingly contradictory responses. in vivo studies to understand the actions of menthol on the vasculature. Historic records about the application of menthol-containing herbs or products as therapeutic agents for cardiovascular disorders are very scarce. Menthol pharmacological properties for the cardiovascular system only became apparent in the 20The transient receptor potential melastatin-related 8 (TRPM8) channels are widely accepted to be the main targets of menthol in different cell types, including neurons and smooth muscle cells of several organs. TRPM8 channels are also implicated in the detection of cool/cold temperatures by afferent A-delta and C nerve fibers  and mentIn the vascular endothelium, extracellular calcium influx leads to calmodulin-mediated activation of nitric oxide synthase (NOS) which increases nitric oxide (NO) synthesis, and consequently leads to VSM relaxation and vasodilation . CalciumMenthol blocks L-type voltage-gated nifedipine-sensitive calcium channels in VSM cells of rat aortic, mesenteric, coronary  and tailThere is also evidence to support that menthol causes vasodilation via suppression of the Rho-associated protein kinase (ROCK) pathway in VSM cells, presumably via TRPM8-dependent and TRPM8-independent mechanisms . The incTransient receptor potential ankyrin transmembrane protein 1 (TRPA1) channels have been identified on endothelial cells of rat arteries , more coTransient receptor potential vanilloid-receptor-related (TRPV) channels 1–4 have been identified in murine and rat arteries and nerves . TRPV3 a2) insofar as its removal by phospholipase enzyme isoforms contributes to this channel’s desensitization  cause skin temperature reduction irrespectively of concentration , which mvia intradermal microdialysis, menthol is directly delivered to the dermal microvascular networks, therefore reducing the contribution of heat-conservation responses initiated by cold perception in the epidermal sensory fibers. Studies that investigated the vascular response with this technique also benefited from the use of lactated Ringer’s solution as a solvent instead of ethanol  endothelial TRPM8 activation leading to an increase in NO release ; (2) VSM L-type voltage-gated calcium channel blockade; (3) RhoA/ROCK pathway suppression. Several other mechanisms have been proposed to contribute to this response, although they need to be experimentally tested. For example, menthol co-activation of TRPA1 and TRPV channels in sensory afferent fibers, endothelial and/or VSM cells have been proposed , even thParenteral and oral routes have also suggested a considerable BP lowering capacity for menthol. The anti-hypertensive potential of menthol was first recognized by Rakieten in 1957 with the intravenous administration to lower BP in cats and rabbits . When adin vivo seems to depend on whether the vascular territory under analysis has been directly provoked with menthol or is located deep/distant to the application site. Menthol increases perfusion of directly provoked skin regions due to a complex interplay of increased NO, EDHFs and sensory nerve responses. In non-provoked vascular beds menthol induces vasoconstriction which might be attributed to heat-conservation sympathetically-mediated vasoconstriction, although an increase in tissue evaporative heat loss due the formulation ethanol may also play a role. There is increasing evidence that several of menthol’s cellular targets are involved in cardiovascular diseases, such as hypertension. Thus menthol and pharmacologically-similar drugs can play important preventive and therapeutic roles, which merits further investigation.Menthol is a versatile molecule with complex actions on the vasculature which remain far from clear. Several cellular targets have been identified in both endothelial and VSM cells as well as in afferent nerve fibers involving a crosstalk between multiple signaling pathways. Menthol is known to activate TRPM8 channels in the endothelium, VSM and vascular afferent nerve fibers, while also blocking VSM L-type calcium channels. Several other menthol targets have been identified in non-vascular tissues including TRPA1, TRPV1 and TRPV3 channels, but whether menthol also acts on vasculature via these channels is still undetermined. Menthol vascular action HS contributed literature research and manuscript editing.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As tumors grow, cancer cells must overcome the normal signals designed to keep cell growth in check. Most cancer cells do this by turning off proteins that prevent growth or turning on proteins that stimulate growth through mutation but also through changes in the levels of these proteins inside cells. This review article summarizes recent research that suggests the acidity or basicity (pH) of the environment inside cancer cells may allow cancer cells to specifically stabilize proteins that help them grow and remove proteins that induce cell death. We also discuss new research tools that allow us to measure and manipulate pH in cells to better understand the role pH plays in enhancing cancer growth and progression.An emerging hallmark of cancer cells is dysregulated pH dynamics. Recent work has suggested that dysregulated intracellular pH (pHi) dynamics enable diverse cancer cellular behaviors at the population level, including cell proliferation, cell migration and metastasis, evasion of apoptosis, and metabolic adaptation. However, the molecular mechanisms driving pH-dependent cancer-associated cell behaviors are largely unknown. In this review article, we explore recent literature suggesting pHi dynamics may play a causative role in regulating or reinforcing tumorigenic transcriptional and proteostatic changes at the molecular level, and discuss outcomes on tumorigenesis and tumor heterogeneity. Most of the data we discuss are population-level analyses; lack of single-cell data is driven by a lack of tools to experimentally change pHi with spatiotemporal control. Data is also sparse on how pHi dynamics play out in complex in vivo microenvironments. To address this need, at the end of this review, we cover recent advances for live-cell pHi measurement at single-cell resolution. We also discuss the essential role for tool development in revealing mechanisms by which pHi dynamics drive tumor initiation, progression, and metastasis. Cancer deaths have not significantly decreased, despite improved therapeutics targeting the genetic basis of cancer . CombatiAn emerging hallmark of cancer cells is dysregulated pH dynamics producing an increased intracellular pH pHi > 7.4) and decreased extracellular pH (pHe < 7.2) relative to normal epithelial cells  A. This r.4 and de+-ATPase  raise iV-ATPase . FurtherBox 1+-ATPases (V-ATPases) represent afamily of ATP-dependent proton pumps that contribute to organellar andcytosolic pH. The primary role of V-ATPases is in regulating lysosomal pH,where they establish an acidic luminal pH to enable optimal hydrolaseactivity. More broadly, V-ATPase involvement in the endolysosomal pathway hasclear implications for altering signal transduction and developingchemoresistance in cancer that has previously been reviewed [Vacuolar Hreviewed . Recent reviewed . This inreviewed . The comRecent work has suggested that dysregulated pHi dynamics enable diverse cancer cellular behaviors at the population level including cell proliferation, cell migration and metastasis, evasion of apoptosis, and metabolic adaptation. These pH-associated cancer cell behaviors have been summarized in several recent review articles ,17,18. FIn this review article, we will describe and explore recent literature that suggests pHi dynamics may play a causative role in regulating or reinforcing tumorigenic transcriptional and proteostatic changes at the molecular level .Most of the data discussed herein are population-level analyses; the lack of single-cell data is primarily driven by the lack of tools to experimentally change pHi with spatiotemporal control. Data are also sparse on how pHi dynamics play out in complex in vivo microenvironments. To address this need, at the end of this review, we cover some recent advances for live-cell pHi measurement at single-cell resolution. We also discuss the necessary and essential role that tool development will play in further characterizing the mechanistic role pHi dynamics play in tumor initiation, progression, and metastasis .During cancer development, cells undergo significant molecular and phenotypic changes in response to their ever-changing environment. This “cellular plasticity” can be driven by mutations, epigenetic regulation, and transcriptional regulation. Transcriptional regulation is orchestrated by myriad factors including transcription factors (TFs) ,33, co-rNotably, it has been shown that pHe ,42 and pDynamic pH can also directly regulate transcription factors to drive phenotypic plasticity in cancer. For example, acidic culture conditions can induce nuclear localization of the Sterol Regulatory Element-binding protein 2 (SREB2), driving the transcription of twelve pH-responsive genes including acetyl-CoA synthetase 2 (ACSS2), 3-hydroxy-3-methylglutaryl-CoA synthase 1(HMGCS1), farnesyl diphosphate farnesyltransferase (FDFT1), and low density lipoprotein receptor (LDLR)  that conIn addition to extracellular pH, pHi has been shown to control the subcellular localization of the transcriptional effector, Smad5 . Fang anIt has been shown that some somatic mutations may confer a pH-sensitivity to the mutated protein . ImportaAdditionally, future work investigating point mutations in other transcription factors may reveal more examples of adaptive gain in pH-sensitive DNA binding. One such possibility is point mutations in the transcription factor FOXP2, that have been shown to play a role in stabilization of DNA binding, where loss of Arg leads to disease . InteresTaken together, these examples demonstrate that disrupted pH dynamics can drive transcription factor localization or activity and lead to alterations in transcript abundance. Moreover, these data suggest environmental cues such as pH could be key drivers of the heterogeneous acquisition of adaptive fitness advantages in cancer cells. Recent work by Persi et al. has determined that pH-dependent vulnerabilities exist in cancer cell metabolism , suggestAs cancer progresses, heterogeneous subpopulations of cancer cells arise with different phenotypic  and moleWhile hypoxia  and metaIn addition to pHi potentially driving microenvironment heterogeneity, recent work shows that pHi may itself be heterogeneous. For example, when cells were selected at distinct pHi levels, their resulting daughter cells had heterogeneous pHi . This suFollowing the central dogma of biology, the amount of mRNA transcripts should be directly correlated to the amount of protein it encodes. However, studies of ovarian , colorecDysregulation of the synthesis-degradation axis alters protein abundance and can confer fitness advantages to cells . FitMany pHi-dependent tumorigenic behaviors, such as proliferation, epithelial-to-mesenchymal transition (EMT), and metabolic reprogramming have been shown to be driven by proteostatic changes. For example, stabilization of the oncogene Myoferlin  enables Proteasome-mediated degradation is regulated by phosphorylation then ubiquitination, where ubiquitin targets proteins for degradation. Highlighting the importance of degradation motif recognition, a recent analysis of new and known degradation motifs determined that nearly 10% of driver mutations in cancer occur within degrons of substrates or substrate recognition interfaces of E3 ligases . Thus, aThe role of pHi dynamics in protein stability has been exemplified in a recent study showing β-catenin functioning as a pH-sensor with decreased stability at increased pHi . A histiOverabundance of proteins like Myc and Cyclin E are associated with cancer cell proliferation and cell cycle progression, respectively. Additionally, Ras and Raf are members of the mitogen-activated protein kinase (MAPK) pathway and constitutive activation results in uncontrolled growth signaling in cancer. However, the abundance of these proteins has never been studied in the context of pHi. We next discuss each protein individually, highlighting key experiments that suggest pH-dependent protein stability may enable their tumorigenic function.Myc is a cancer-associated transcription factor that is targeted for proteasome-mediated degradation by the E3 ligase, Fbxw7. This E3 ligase requires three arginine residues  for substrate recognition and binding , with 29Cyclin E is a kinase that progresses cells through the G1-S transition into DNA synthesis, where dysregulated abundance may result in genomic instability. Previous reports showed constitutive activation of Ras or Raf increased cyclin E abundance, suggesting MAPK activation is sufficient to prevent ubiquitination of cyclin E . The autFraming the results from previous studies in the context of pH dynamics reveals a potential role for pH in proteostasis-mediated tumorigenesis. For example, a mutation in either an oncogene (Myc) or tumor suppressor (Fbxw7) may invoke pH-sensitivity that was previously overlooked in protein abundance dynamics. Alternatively, activation of NHE1 and resulting intracellular alkalinization may increase kinase activity to regulate protein degradation pathways and reinforce tumorigenesis. Degradation-associated signaling cues and kinase activation may also be mediated through pHi changes, as we propose for Cyclin E, where activation of NHE1 and resulting intracellular alkalinization increases kinase activity to dysregulate protein degradation pathways and reinforce tumorigenesis. However, it is still unclear how specific molecular events and microenvironment cues may drive dysregulation of proteostasis. While studies involving β-catenin have highlighted pHi as a regulator of protein degradation, more work needs to be done to validate the other examples—Myc and Cyclin E—proposed here.Correlations can be made between pHi and transcriptional and proteostatic changes, but we lack studies that conclusively show whether pHi is a common driver of these changes that lead to the acquisition of cancer fitness advantages. Importantly, initiating these mechanistic studies requires improved tools to accurately measure and specifically manipulate pHi for transcriptomic or proteomic analysis. In this section of the review, we will discuss the tools and techniques used to measure and manipulate pHi in current studies, as well as the improvements that need to be made to current methodologies to reveal the complex role of pHi dynamics on transcriptional and proteomic changes in cancer.The ability to accurately measure pH inside living cells is critical to understanding pHi dynamics during cellular processes and the effect they have on cellular behaviors. Doing so requires tools that can measure absolute pH values with high spatial and temporal resolution. Ideally, these tools can be used to measure live, single-cell pHi with minimal damage to the cell. Please refer to Early methods of pHi measurement include the use of patch clamp techniques to analyze pHi . While tThe advent of molecular tools to measure pH in living cells helped fill this gap. These tools are capable of measuring pH with high spatiotemporal resolution, decreased technical complexity, and increased reproducibility. There are several groups of molecular tools that have been developed for the measurement of pHi including fluorescent dyes , ionic lFluorescent pH-sensitive dyes such as the green fluorescent BCECF -5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester) , and theWork with other synthetic reporters has recently made improvements such that they are more compatible with long time-scale in vivo work. For example, indole heptamethine cyanine dyes represent another class of pH-sensitive dyes that are useful for tumor and tissue measurements due to their near infrared fluorescence properties; however, they suffer from poor stability and dim fluorescence . Ionic lDeveloping fluorescent, protein-based tools to measure pHi is transformative because it allows for stable expression of a sensor that can be easily targeted to subcellular compartments. Most green fluorescent proteins (GFPs) are natively sensitive to pH with pKas around 6.0 . The fluTo combat some of the issues with pHluorin, red-shifted fluorescent proteins (RFP) that function as pH biosensors have been recently developed, including pHred , pHuji , and morThe ability to manipulate pHi allows us to determine the role pH plays as a potential initiator or driver of tumorigenic cell behaviors. To reiterate, the ideal pHi manipulation tool must have high spatial and temporal resolution, minimal off-target or cell-toxic effects, should be capable of cue-dependent responses and be reversible.Some of the more recently developed tools focus on various sources of cue-dependent pH manipulations. NMR instruments have been shown to be capable of inducing pH changes in solution . Some exCurrent techniques for manipulating pHi often involve disrupting native cellular pH homeostasis mechanisms, such as knock-down or inhibition of specific proton transporters. However, these methods suffer from widespread off-target effects and poor spatial and temporal resolution. Temporal resolution could be improved with transient single-cell knockdown and both spatial and temporal resolution could be improved with photo-uncaging of inhibitors or pH alkanization agents.More promising spatiotemporal pHi manipulation tool development can be seen in optogenetic tools. While light-activated proton pumps, such as archaerhodopsin, have mainly been used to silence neuronal firing, they have recently been used to alter pHi in cells to monitor gap junction connectivity . HoweverCurrent tools to measure  and manipulate  pHi have been useful in establishing our understanding of how pHi functions within the realm of cancer development and progression. However, there is a need for improved tools capable of measuring pHi inside of tissue and animals accurately and with low toxicity. Such tools will allow the study of pHi dynamics of cancer cells within the native tumor microenvironment with minimal perturbation. New tools for pHi manipulation with decreased cytotoxic effects, specific pHi manipulation, and precise control will improve studies to investigate pHi as a driving force in cancer.It is clear that phenotypic and genotypic heterogeneity function in concert with microenvironment pressures to create a perfect storm of cancer survival, metastasis, resistance, and evolution. Key to improving patient outcomes is a better understanding of molecular mechanisms driving these cancer hallmarks. Cancer genomics is advancing at an incredible rate, with innovations in single-cell DNA sequencing , four-diThe work summarized in this review suggest the tantalizing possibility that tumor pHi may be a biomarker of more complex and difficult to measure markers of tumor heterogeneity such as cancer metabolism, cancer stem cells, or epithelial vs. mesenchymal phenotypes. The work described above suggests that the cell biological and biochemical effects of increased pHi in cancer correlate with and perhaps reinforce transcriptional and proteostatic changes associated with cancer. However, several recent papers that note off-target effects of key pHi manipulating drugs ,131 haveAs this review shows, our current understanding of pH-sensitive proteins and cell behaviors has been built from decades of slow and careful one-by-one studies. With better tools, we can validate the literature results as well as access previously intractable experiments that explore the role of pHi in regulating global transcriptomic and proteostatic changes. This will allow us to identify pH-sensitive nodes in these cancer-associated pathways that might be therapeutically targetable for limiting tumor progression. Furthermore, quantitative pHi measurements across cancer model systems could alone revolutionize the way clinicians think about targeting pHi-dependent cancer fitness advantages.


Histological and epidemiological data suggest that increased signal intensity at the proximal patellar tendon on magnetic resonance imaging is a response to tendon loading. As patellofemoral geometry is a mediator of loading, we examined the association between patellofemoral geometry and the prevalence of increased signal intensity at the patellar tendon in community-based middle-aged adults.Two hundred-one adults aged 25–60 years in a study of obesity and musculoskeletal health had the patellar tendon assessed from magnetic resonance imaging. Increased signal intensity at the proximal patellar tendon was defined as hyper-intense regions of characteristic pattern, size and distribution on both T1- and T2-weighted sequences. Indices of patellofemoral geometry, including Insall-Salvati ratio, patellofemoral congruence angle, sulcus angle, and lateral condyle-patella angle, were measured from magnetic resonance imaging using validated methods. Binary logistic regression was used to examine the association between patellofemoral geometrical indices and the prevalence of increased signal intensity at the patellar tendon.p = 0.02), indicative of a higher-riding patella, and a larger patellofemoral congruence angle , indicating a more laterally placed patella, were associated with reduced odds of increased signal intensity at the patellar tendon. Sulcus angle and lateral condyle-patella angle were not significantly associated with the odds of increased signal intensity at the patellar tendon.The prevalence of increased signal intensity at the patellar tendon was 37.3%. A greater Insall-Salvati ratio (odds ratio 0.80, 95% confidence interval 0.66–0.97 per 0.1 change in the ratio, In community-based asymptomatic middle-aged adults, increased signal intensity at the patellar tendon was common and associated with Insall-Salvati ratio and patellofemoral congruence angle, suggesting a biomechanical mechanism. Such work is likely to inform tissue engineering and cell regeneration approaches to improving outcomes in those with tendon pathology. The structure-function relationships in articular cartilage and subchondral bone have been widely investigated, but relatively little attention has been given to tendons. The increasing interest and effort in cell regeneration therapy and tissue engineering to promote tendon repair and create artificial tendons , 2 highlMagnetic resonance imaging (MRI) enables non-invasive assessment of joint structures with signal alterations reflecting tissue composition. Histological examination of increased signal intensity of the proximal patellar tendon on T1-weighted and fluid-sensitive MRI have been shown to be due to invaginated adipose tissue, vessels, and perivascular connective tissue . We havePatellofemoral geometry has an important role on the direction and magnitude of forces acting on the patellofemoral joint and patellofemoral alignment may affect load distribution to the extensor mechanism, thus altering the distribution of strain within the patellar tendon upon quadriceps contraction , 7. GiveTwo hundred and fifty participants, aged 25–60 years and across the spectrum from normal weight to obese, were recruited into a study investigating the effect of obesity and weight loss on knee health. They were recruited via advertising in the local media, hospital waiting rooms, community weight loss organisations, and private weight loss and obesity clinics . Partici2). Strenuous physical activity was assessed as previously described  and greater Insall-Salvati ratio  compared with those with patellar tendon assessment.Among the 250 participants, increased signal intensity at the patellar tendon could be assessed in 201 (80.4%) participants, with 49 participants without the assessment due to the unavailability of particular T2-weighted coronal images to confirm the presence of increased signal intensity at the patellar tendon. These 49 participants had higher BMI [37.8 (SD 9.7) vs. 33.2 (SD 9.3) kg/mn = 75, 37.3%) were more likely to be male , had lower Insall-Salvati ratio  and smaller patellofemoral congruence angle , compared with those without increased signal intensity at the patellar tendon  remained significantly associated with decreased odds of increased signal intensity at the patellar tendon. There was no significant association for sulcus angle or lateral condyle-patella angle in either univariable or multivariable analysis , were associated with reduced odds of increased signal intensity at the proximal patellar tendon.The high prevalence of increased signal intensity at the proximal patellar tendon in our study provides an opportunity to understand patellar tendon health given that this MRI change is known to represent distinct pathological processes of invaginated adipose tissue, vessels, and perivascular connective tissue . A high-The reduced odds of increased signal intensity at the patellar tendon related to a larger patellofemoral congruence angle observed in our study is supported by our previous study reporting an association between greater vastus medialis cross-sectional area and higher prevalence of increased signal intensity at the patellar tendon . GreaterLimitations of our study include the cross-sectional analyses for the association between patellofemoral geometry and increased signal intensity at the proximal patellar tendon, and thus the causality cannot be examined. While highly reproducible, the increased signal intensity at the proximal patellar tendon detected from MRI may not correlate with clinical disease , 20. HowOur study showed a high prevalence of MRI-detected increased signal intensity at the proximal patellar tendon in a sample of community-based middle-aged asymptomatic adults across a range of BMI. The lower odds of increased signal intensity at the patellar tendon in relation to greater Insall-Salvati ratio and patellofemoral congruence angle supports biomechanical mechanisms. Such work is likely to inform tissue engineering and cell regeneration approaches to improving patellar tendon morphology and outcomes in those with tendon pathology by targeting patellofemoral geometry to alter patellofemoral joint mechanisms.


Suicide gene therapy has represented an experimental cancer treatment modality for nearly 40 years. Among the various cancers experimentally treated by suicide gene therapy, high-grade gliomas have been the most prominent both in preclinical and clinical settings. Failure of a number of promising suicide gene therapy strategies in the clinic pointed toward a bleak future of this approach for the treatment of high-grade gliomas. Nevertheless, the development of new vectors and suicide genes, better prodrugs, more efficient delivery systems, and new combinatorial strategies represent active research areas that may eventually lead to better efficacy of suicide gene therapy. These trends are evident by the current increasing focus on suicide gene therapy for high-grade glioma treatment both in the laboratory and in the clinic. In this review, we give an overview of different suicide gene therapy approaches for glioma treatment and discuss clinical trials, delivery issues, and immune responses. The urgent need for novel and more efficient treatment strategies has produced various novel molecularly targeted therapeutic options that have been tested in clinical trials alongside the traditional chemotherapy/radiation approaches.2 However, the outcomes have mostly fallen short of expectations.3 Another experimental treatment modality that has persistently been tested in HGG patients is suicide gene therapy (SGT) or also known as gene-directed enzyme prodrug therapy. The initial successful proof of concept in animal models4 and small-scale clinical trials5 could not be replicated in a large-scale trial6 generating a negative wave against further development of SGT. However, continued research has promoted further improvements of this strategy resulting in a more tailored therapeutic avenue for HGG. SGT is a multi-componential approach (High-grade gliomas (HGGs), collectively known as WHO grade III and IV primary brain tumors, belong to the most deadly and incurable group of cancers.approach  and thusSGT represents the most common form of gene therapy used to treat HGGs in both preclinical and clinical settings. From a theoretical perspective, SGT is a two-step treatment modality for solid tumors : the fir7 (9 adopted this concept for cancer treatment by showing that delivery of cytosine deaminase (CD) from Escherichia coli followed by 5-fluorocytosine (5FC) administration leads to a significant reduction of tumor burden in a syngeneic EA285 rat glioma model. Moolten et al.11 contemporarily introduced SGT based on thymidine kinase (TK) from HSV for cancer treatment , rendered the cells more sensitive to acyclovir (ACV)7 . Nishiyareatment . CD and ablished .12 Therefore, 5FU itself has been used as a potent anticancer chemotherapeutic agent for many years, but off-target toxicity has limited its direct applicability.13 Two different CD proteins have been adopted for developing the SGT system with CD/5FC: bacterial CD  and yeast CD . Although bCD and yCD bear little homology, they catalyze cytosine and 5FC in a similar fashion, albeit with different efficiency. yCD has a significantly lower Km and a higher Vmax for 5FC than bCD13; however, yCD exhibits thermal instability. The enzyme works optimally at around 26°C and loses activity as temperature rises.13 This shortcoming has been mended by rational protein engineering that involves alteration of 3 amino acids in the yCD gene.14 Currently, both bCD and the recombinant yCD are being used for HGG treatment.16CD, an enzyme found in bacteria and lower eukaryotes , is involved in microbial pyrimidine metabolism and deaminates 5FC  into 5-fluorouracil (5FU). 5FU is a pyrimidine analog that directly inhibits nucleic acid synthesis due to misincorporation instead of uracil or thymine. 5FU can also be catalyzed by cellular enzymes into fluorodeoxyuridine monophosphate that can interfere with DNA metabolism by binding thymidylate synthase.TK gene that is evolutionarily and functionally different than the human thymidine kinases (hTKs).17 Compared to hTKs, HSV-TK more efficiently catalyzes various prodrugs  producing mono-phosphorylated nucleoside analogs that are further phosphorylated by cellular kinases.17 The resulting triphosphorylated analogs are incorporated into DNA strands during replication and cause strand abrogation leading to cell death of actively proliferating cells. Importantly, the analogs  are not efficiently recognized by the hTKs preventing toxicity for normal cells. As a result, HSV-TK acts as a suicide gene upon prodrug exposure without any major interference of the hTKs. Various purine and pyrimidine analogs are compatible with HSV-TK SGT such as ganciclovir (GCV), ACV, and brivudin (BVDU).19 BVDU is an efficient substrate of HSV-TK and a potent inducer of cell death,20 but exhibits poor BE.21 GCV is a better substrate for HSV-TK compared with ACV and exhibits a greater BE compared to either ACV or BVDU.22 Valganciclovir , an oral analog of GCV, has recently been shown to be suitable for long-term treatment in a GBM xenograft model.23HSV encodes a 24 and presence of cryptic sites leading to anomalous transcription25 or splicing.26 Such limitations can be overcome by optimizing sequences of the HSV-TK gene,27 and a novel mutant with superior functionality has been developed to be used for treatment of experimental HGG.29The wild-type HSV-TK suffers from a few shortcomings: higher affinity toward its natural substrate endogenous thymidine (dT) compared to GCV10 γ-retroviral vectors (RVs) and later adenoviral vectors (AdVs) were employed to deliver suicide genes into tumors.30 Although these vectors were effective in glioma animal models and to some extent in early-phase clinical trials, results from a larger phase III trials were disappointing.31 Treatment failure was mostly attributed to various shortcomings of the viral vectors indicating that more efficient vector systems need to be developed to harness the power of SGT for cancer treatment. Thus, per today a wide variety of vectors derived from different viral backbones are used for SGT  therapy demonstrated a significant therapeutic benefit in experimental glioma models by several independent investigators34 and also in small-scale HGG clinical trials.35–37 No serious adverse effects were observed, which is an important finding since AdVs had caused death of a patient in a trial for ornithine transcarbamylase gene therapy.38 Thus, a phase III clinical trial (known as ASPECT) was performed to evaluate the potential therapeutic benefit of AdV-TK/GCV therapy as an adjuvant treatment arm to standard of care .31 While ASPECT showed significant improvement in “time-to-re-intervention” compared to standard care, unfortunately the study failed to show a significant effect on overall survival. Although there was a stronger treatment effect in patients with non-methylated MGMT , the results did not satisfy the European Medicines Agency (EMA) and thus the marketing request was denied. Several theoretical reasons could be attributed to the failure of the ASPECT trial. Firstly, it is possible that the interpretation of the therapeutic outcome of the SGT was compromised by the lack of universal standard care protocols  across all institutions.39 Secondly, the therapy itself might not have been effective enough. Despite improved transduction capability, AdVs suffer from a major drawback by the lack of long-term expression of transgenes. Unlike RVs, AdVs do not integrate into the host genome and thus transgenes are only expressed transiently for up to a few weeks.40 In this context, we have recently shown that short-term SGT can result in treatment-escape and that long-term suicide gene activity can improve treatment efficacy.23 While the problem of short-term gene expression can be somewhat mitigated in the CNS where the transgene expression may be detected for at least up to 3 months,18 the episomal maintenance of AdVs still causes the transgene expression to be reduced following cell divisions that would likely lead to compromised efficacy during tumor expansion.The failure of RV-mediated SGT in a phase III clinical trial42 Other combination strategies such as concomitant TMZ or Nivolumab (checkpoint inhibitor) are also being pursued in different clinical trials at this point , AdV-mediated SGT is still pursued in both laboratories and the clinic with different combinatorial HGG treatment strategies. In this context, concomitant radiotherapy with AdV-TK/ACV has recently been tested in clinical trials showing notable improvement in survival outcomes.is point . Anotheris point .43,4451 Since RRVs exhibit a non-lytic life cycle, the therapy is mostly dependent on the suicide gene activity which could be achieved by using CD or viral TK.51 While RRVs in general transduce only dividing cells, the high-transduction ability is conferred by the replicative nature of the vectors. Most importantly, RRV replication is restricted to glioma cells in vivo due to the post-mitotic state of most normal cells within the CNS.51 By using a recombinant yCD gene,47 several clinical trials funded by Tocagen, Inc., were performed demonstrating safety as well as encouraging results compared to external lomustine-treated cohorts.52 These positive indications resulted in the designation of breakthrough and PRIME status for HGG treatment by FDA and EMA, respectively. The clinical studies took advantage of next-generation sequencing technologies revealing some interesting aspects of the therapy in relation to associated prognostic factors. For example, an important discovery was made by identifying a transcriptomic signature, termed survival-related neuronal subtype (SRNS), that is associated with Toca 511/5FC-mediated survival.51 Interestingly, the SRNS signature shows functional similarities with the TCGA neural subtype and thus patients who exhibited SRNS  benefitted most from Toca 511/5FC. Aside from SRNS, some other prognostic factors were also identified including a tentative identification of Isocitrate dehydrogenase 1 (IDH1) mutation as a positive prognostic factor.52 Furthermore, the study revealed that the activity of CD/5FC, similar to HSV-TK/GCV, is independent of patient MGMT status. The preliminary success warranted a phase III clinical trial (NCT02598011) which has been performed in 403 HGG patients. Recently Tocagen, Inc., made a press release announcing that the phase III trial unfortunately failed to meet the study endpoints.53 While detailed results are currently unavailable, the failure once again reveals the tremendous challenge of treating HGGs. Further improvement related to this treatment strategy may most likely depend on combinatorial approaches with other treatment modalities.The major drawbacks of RVs and AdVs, namely, low transduction rate and episomal nature may be circumvented by using replicating retroviral vectors (RRVs). RRVs of non-primate origin have been reported to efficiently transduce glioma cells and thus refocused the translational attention of SGT involving γ-RVs.48 where a significant fraction of non-dividing tumor cells is present. These resting glioma cells could not be targeted by RVs neither in vitro nor in vivo. LVs are most frequently pseudotyped with the glycoprotein (GP) of vesicular stomatitis virus (VSV-G). However, GPs from other viruses can also be used. We have shown that LVs pseudotyped with the GPs of the lymphocytic choriomeningitis virus transduce glioma cells more specifically compared to VSV-G pseudotyped vectors which have a strong tropism for neurons.54 To investigate therapeutic efficacy, LV-based HSV-TK/GCV treatment was tested in a patient-derived GBM xenograft model and subsequently complete, albeit temporary, tumor remission after GCV administration was observed.48 In this study, a fraction of tumor cells from recurrent tumors still expressed the suicide gene indicating that short-term prodrug administration (2–3 weeks), which is currently standard in clinical trials, is not sufficient to achieve the maximum treatment effect. This hypothesis was confirmed in a follow-up study showing that long-term administration of valGCV, a prodrug tailored for oral application, increased the treatment effect compared to short-term GCV treatment.23 Due to unrestricted transducing potential, LVs, in particular those pseudotyped with VSV-G, can also transduce normal post-mitotic brain cells, however, without toxicity even when using HSV-TK/GCV.55 On the contrary, normal brain cells expressing HSV-TK contribute in eliminating glioma cells through BE.49 LV-based SGT has not yet been tested in clinical trials; however, the various auspicious features observed in the most clinically relevant GBM models strongly warrant clinical investigation.Lentiviral vectors (LVs), developed as a spin-off from HIV research, serve as one of the most popular vector systems in gene therapy. In contrast to the aforementioned vectors, LVs offer 2 unique features that are very important for SGT toward HGGs: transduction capability in quiescent cells and long-term gene expression. Similar to all retroviruses, lentiviruses integrate the provirus into host-cell genomes. However, unlike γ-retroviruses, lentiviruses are equipped with active nuclear transport machinery leading to genome integration independent of mitosis. LVs, availing this mechanism, can transduce quiescent glioma cells much more efficiently than RVs, as shown in biopsy-based glioma spheroids,50 In general, these cells show an intrinsic migratory capacity and exhibit exceptional tropism toward pathological conditions including neoplastic lesions in the CNS.56 The tumor-tropic property of these cells has its origin in the bona fide regenerative and reparative roles in cellular homeostasis which also relates to the sensing of various pro-tumorigenic signals such as angiogenesis, hypoxia, inflammatory signals, etc..57–60 Furthermore, these cells survive in vivo engraftment  for a certain time period due to low or undetectable MHC expression56 and do not form neoplastic lesions indicating a high safety profile.56 Cell-mediated tumor-killing activity solely depends on the BE and thus an SGT system with high bystander efficiency is a prerequisite.61 NSCs were the first type of cells used as a vector for SGT. The delivery potential of NSCs for SGT of glioma was first reported by using an immortalized murine NSC line which was retrovirally transduced with bCD. The engrafted NSCs migrated in a glioma-specific manner, both ipsilaterally and contralaterally, and were able to kill tumor cells upon 5FC administration.15 An important issue for clinical translation of NSC-mediated SGT (or any cell-based system for that matter) is to choose between an autologous and allogeneic source. Ideally an autologous source of NSC will be preferential based on immune escape. Although NSCs normally show low levels of MHC expression, there exists an immunogenic potential62 that would eventually promote the clearing of allogeneic NSCs within week(s).56 This issue can be partially circumvented by using immunosuppressive drugs; however, this may thwart anticancer immune responses and interfere with the overall treatment efficacy. Furthermore, the use of autologous NSCs is associated with several logistic shortcomings such as lack of adequate source as well as long-term culture for expansion to large cell numbers for clinical application. Cellular reprogramming technologies such as induced pluripotent stem cells are currently being pursued to obtain sufficient cell numbers of autologous NSCs.64 In contrast, the use of allogeneic NSCs offers several advantages over autologous NSCs in terms of time, cost, scalability, and standardization procedures.57 To date, two different NSC-mediated SGTs have been pursued in clinical studies and both are based on an immortalized allogeneic human NSC line known as HB1.F3.66 The first trial involved treatment with a bCD-modified HB1.F3 cell line in order to evaluate initial safety and feasibility.67 The NSCs were observed to migrate to distant tumor sites in the HGG patients and initial safety was demonstrated. As a result, a phase I trial with 18 patients has been started which will be completed soon  and mesenchymal stem cells (MSCs) are the major sources for this strategy. Recently, olfactory ensheathing cells have also been shown to be an efficient vector for SGT.ted soon . The sected soon .68 possess an intrinsic migratory capacity toward pathological lesions similar to NSCs and in this regard no substantial differences have been found between these two cell types.69 In addition, MSCs can be derived from various tissues and organs such as bone marrow, adipose tissue, umbilical cord blood, and placenta68 offering better accessibility for procurement compared to NSCs. Furthermore, MSCs can be easily expanded to high cell numbers for clinical application. Thus, the accessibility and also scalability of MSCs can provide advantages over NSCs for treatment application. The first SGT approach using MSCs for HGG treatment was reported by Miletic et al.70 in an orthotopic, syngeneic rat glioma model. The study showed a substantial treatment effect of intratumorally injected MSCs termed bone-marrow-derived tumor-infiltrating cells expressing HSV-TK following prodrug treatment. The direct tumor cell killing by MSCs was mediated through BE, while an immune response with infiltration of T cells and NK cells was detected in the treated tumors, which may have contributed to the treatment effect.70 Since then a number of preclinical studies have been published.71 However, no clinical trial has been performed yet for HGG treatment.MSCs72 but challenging due to potential off-target transduction in non-CNS tissues and/or insufficient bioavailability in the CNS. Intranasal delivery of cell-based vectors has also been pursued successfully in preclinical models.73 However, intracranial injection has been the most popular mode of administration and for the majority of SGT trials so far vectors have been injected directly into the resection cavity after surgery using multiple injections.51 This method of application is suboptimal as the tissue around the resection cavity is very heterogeneous containing either diffusely infiltrating tumor cells or reactive brain tissue. Thus, there is no control of how much tumor tissue is reached with this method, which might also partly explain the failure of clinical trials. There is a huge interpatient variation concerning injection efficacy and the amount of target tissue reached, which makes interpretation of clinical trial data extremely difficult. CED is a sophisticated delivery method into solid tissue that has been developed in particular for the brain and also brain tumors.74 CED is applied through stereotactically placed catheters that are connected to a micropump maintaining a continuous low-pressure flow into the tissue. This method has substantially increased the amount of tissue that can be targeted and thus is frequently used to inject vectors or drugs into the brain.74 Regarding the treatment of brain tumors, this method is optimized for application into solid tumor tissue, however not into a resection cavity after surgery. The problem that emerges from here is that primary tumors are usually treated by neurosurgery that is the standard of care. Thus, at this point, the only choice in order to implement CED into future clinical trials is to inject the vectors either into recurrent tumors or inoperable primary tumors.Delivery of gene therapeutic products into brain tumors is an important issue that is critically discussed in the field, however still lacks optimal solutions. Systemic delivery of viral vectors is feasible75 AdVs are capable of eliciting an acute immune response including secretion of several proinflammatory cytokines.18 However, vector-induced immune responses can be a double-edged sword with either limiting transgene delivery and thereby impeding treatment efficacy or in contrast potentially breaking the immune tolerance of the glioma microenvironment76 and thereby enhancing the treatment effect of SGT.In general, potential antitumor immune responses mediated by SGT can originate from its different modules such as the type of vector, the cell death mechanism following suicide activity, the type of prodrug, the immune microenvironment of the tumor, and any additional treatment regimen. Different types of vector systems can have a variable impact on the immune system. While RRVs, LVs, and NSCs are not highly immunogenic in nature,77 Glioma cells also undergo HSV-TK/prodrug-mediated cell death via apoptosis.79 The immunogenicity of apoptosis in general is controversial and not yet fully explored, in particular not in the context of SGT. Considering the plastic nature of leukocytes and their dichotomous role in antitumor IR,81 more studies are warranted to unravel the exact nature of HSV-TK/GCV-mediated cell death from an immunogenic point of view.The mechanism of HSV-TK/prodrug-mediated cell death can be variable involving necrotic and immunogenic cell death (ICD) in melanoma cells, but apoptotic and non-ICD in colorectal cancer cells.30 and clinical settings.82 Still, in glioma, the antitumor IR elicited by AdV-HSV-TK/GCV therapy is often not strong enough without additional immunostimulatory strategies.43 When boosted by the co-expression of Flt3L or treatment with checkpoint inhibitors such as anti-PD1 antibodies, AdV-HSV-TK/GCV elicits a more robust antitumor IR.83–85 The co-expression of Flt3L has been shown to recruit dendritic cells to the tumor microenvironment and thereby increase antigen presentation and subsequently T-cell infiltration and activation in glioma animal models.83 This strategy is currently being investigated in phase I clinical trial  which contain such quiescent cells and are considered as the new standard for preclinical studies in gliomas.91 Another important issue is the delivery of the vectors as discussed above. CED could overcome the poor distribution of viral vectors; however, the presence of a solid tumor mass instead of a resection cavity is clearly preferred. Preclinical studies should be performed to test this hypothesis.Over the last decade(s), new developments in SGT have emerged, in particular new vectors and suicide genes with improved affinity to prodrugs . There a93 ICD might thus be a compulsory prerequisite for the engagement of an effective antitumor IR. More fundamental research is needed to identify the intrinsic nature of cell death, namely, immunogenic or immunosuppressive by the SGT systems in GSCs. If one SGT system fails to elicit strong ICD in GSCs (or in primary glioma cells), novel strategies or combinations could be designed to reroute the cell death mechanism. Unfortunately, the syngeneic animal models for glioma available today remain a serious drawback in this regard, because the immune microenvironment in these models differs substantially in composition from the one observed in HGG patients. The use of GSCs in humanized rodent models in this context may create valuable new information. Thus, a number of different preclinical models should be adopted to further develop SGT in the future and to achieve a better clinical translation procedure (The immunosuppressive glioma microenvironment represents another clinical challenge. Although there are indications that the SGT-induced cell death is immunogenic, this has not been tested in detail, especially not in gliomas. Here, a lot can be learned from oncolytic viruses where an antitumor immune response is promoted by the vectors’ ability to induce ICD through oncolysis and thereby recruit an efficient antitumor IR.rocedure .Combinatorial approaches using SGT with co-expression of immune-stimulating cytokines have been performed; however, these approaches might be too unspecific and are mostly directed toward T cells. Yet, T cell infiltration is scarce in glioblastoma, where immunosuppressive microglia and macrophages predominate. To improve future SGT approaches, the changes in the immune microenvironment under SGT should be analyzed more thoroughly. Based on this knowledge, specific shortcomings in the antitumor immune response during SGT could be detected and more targeted approaches could be developed.


Ureteral atresia is the congenital absence of a ureteral opening, resulting in a blind-ended ureter that fails to terminate at the urinary bladder. Consequently, severe hydroureter and hydronephrosis occur ipsilateral to the atresic ureter. However, hydronephrosis contralateral to severe hydroureter, although reported in humans, is not documented in the dog. Additionally, ureteral atresia has not been reported as a cause for lower urinary tract signs directly related to extramural urinary bladder compression. This report aims to describe these unique manifestations of this congenital urinary tract disease, as well as follow-up findings after successful treatment.A 4-month-old male Husky puppy was evaluated for pollakiuria, stranguria, and urine dribbling of 1-month duration. During the physical examination, a mass was palpated in the mid-abdomen. Diagnostic imaging and cystoscopy findings were diagnostic for right-sided ureteral atresia with secondary hydroureter and hydronephrosis. The severe right hydroureter caused lower urinary tract signs and contralateral hydronephrosis secondary to regional compression of the left distal ureter and urinary bladder. A right-sided ureteronephrectomy was performed, resolving the stranguria and pollakiuria. Significant reduction in the contralateral (left) hydronephrosis also occurred.Ureteral atresia should be considered as a differential diagnosis for lower urinary tract signs and/or bilateral hydronephrosis in a young dog. Reporting this case expands our knowledge of congenital lower urinary tract disease and the etiology of their manifestations in dogs. Surgical resolution of the congenital ureteral abnormality can result in preservation of renal function in the contralaterally obstructed kidney. Congenital ureteral anatomic anomalies causing obstructive hydroureter and hydronephrosis are rare in veterinary small animal patients, with various etiologies reported –10. UretIn humans, hydroureter is a reported cause of contralateral ureteral obstruction and bilateral hydronephrosis; however, to the authors’ knowledge, this has not been reported in the dog , 13. HydA 4-month-old, male intact Siberian Husky weighing 11.8 kg was evaluated because of a 1-month history of stranguria, pollakiuria, and urine dribbling. One week prior to presentation, the referring veterinarian prescribed enrofloxacinOn physical examination, the dog had a distended abdomen and palpable mid-abdominal mass. Urination resulted in either a weak broken urine stream or infrequent urine dribbling. Hematology and biochemistry yielded no clinically significant abnormalities . A urine culture was negative for bacterial growth, presumptively due to recent antibiotic administration. Abdominal ultrasonography revealed a 6.2 × 7.8 cm fluid-filled structure consistent with hydronephrosis of the right kidney and severe dilation of the left renal pelvis  , right uAt the follow-up evaluation visit #2), the dog’s lower urinary tract signs persisted of similar severity to visit #1. A recheck ultrasound of the urinary tract revealed progressive hydronephrosis of the right kidney and progressive dilation of the left renal pelvis, right ureter, and left ureter. Suspecting persistent bilateral obstructive disease, an abdominal computed tomography (CT) scan, the dog3 urethrogram revealed an intraluminal filling defect in the pelvic urethra. Flexible urethroscopy performed immediately thereafter using a 2.8 mm ureteroscope4 confirmed mucosal damage and secondary fibrin tags resulting in occlusion of the pelvic urethra, likely iatrogenic from the rigid cystoscopy. An indwelling Foley urinary catheter was placed to allow for urethral healing and clearance of blood clots from the urinary bladder.Twenty-four hours following discharge, the dog re-presented for inability to urinate. Serum creatinine was within reference interval . A red rubber urinary catheter was passed into the urethra with ease and 1100 milliliters of serosanguinous urine containing blood clots was removed from the urinary bladder. A fluoroscopic-assistedTwo days later, a right-sided ureteronephrectomy was performed Fig. . FollowiIntraoperatively, the right ureter was severely distended, resulting in extramural compression and displacement of the urinary bladder Fig. . AdditioGrossly, the right kidney consisted of a thick capsule with no discernable renal tissue and a markedly dilated right ureter that ended in a blind sac. Histologically, the right kidney had scant isolated renal tubules surrounded by haphazardly arranged fibrous tissue and small to moderate numbers of neutrophils and plasma cells. The right ureter had a similar cellular infiltrate as the kidney, with the addition of a diffusely hypertrophied tunica muscularis. The distal blind-ended portion of the ureter contained smooth muscle fibers, confirming ureteral tissue origin. Both gross and microscopic findings were diagnostic of distal ureteral atresia with secondary hydroureter and hydronephrosis. Bacterial cultures of the urine, surgically removed kidney, and fluid within the right hydronephrotic kidney were negative.At a follow-up recheck four months later (visit #3), the dog was urinating a strong urine stream, with no pollakiuria or stranguria noted. Again, serum creatinine remained within reference interval . However, the dog was intermittently dribbling urine, likely the result of the left ectopic ureter. Recheck abdominal ultrasound revealed persistent, but vastly improved left hydronephrosis  and hydroureter  , 15 . Although possible, we feel these explanations for the findings that make this case report novel are less likely for several reasons. First, the urinary tract infection diagnosis is unconvincing, as it is based solely on the presence of leukocytes found on a free-catch urine dipstick sample. Second, the lower urinary tract signs did not improve despite a duration (24 days) of antibiotic therapy that would be considered sufficient to see improvement in clinical signs. Additionally, the negative urine culture one week into the antibiotic treatment indicated that the enrofloxacin therapy was adequate to sterilize the urine, thus appropriately treating any potential bacterial infection present. Third, dilation of the contralateral (left) kidney and ureter worsened on the visit #2 preoperative ultrasound images following 24 days of antibiotic treatment. Finally, the dog displayed no systemic signs  nor had blood work abnormalities  consistent with an ascending infection. In totality, although we cannot exclude the possibility of a concurrent urinary tract infection, these reasons make an infection involving both the lower and upper urinary tract an unlikely contributor to the unique manifestations occurring secondary to the atresic right ureter in this case report.This is the first case report in any animal species of a congenital ureteral anatomic abnormality causing lower urinary tract signs and contralateral ureteral obstruction and hydronephrosis due to extramural compression of the contralateral ureter and urinary bladder. This report expands our understanding of congenital lower urinary tract disease in dogs; albeit rare, this abnormality is an important differential diagnosis in a young dog with bilateral hydronephrosis and lower urinary tract signs, especially if a bacterial urinary tract infection has been ruled out. Furthermore, this case demonstrates that ureteronephrectomy represents a viable long-term treatment option, despite contralateral hydronephrosis being a sequalae of the original anatomic abnormality. Lastly, the diagnostic workup in this case illustrates how CT is becoming incrementally more important for in the diagnosis and treatment of some congenital urogenital diseases.