Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes.

Introduction: The domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing. Methods: Peripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, alpha/beta T cell receptor transcripts and gamma/delta T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5' reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. Results: Unsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and gamma/delta T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated a skewed T-cell receptor alpha gene usage and a restricted T-cell receptor gamma junctional length in CD8+ cytotoxic T cells compared to other alpha/beta T cell subsets. Among myeloid cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster. Discussion: Our study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Homozygous CNP Mutation and Neurodegeneration in Weimaraners: Myelin Abnormalities and Accumulation of Lipofuscin-like Inclusions.

A progressive neurological disorder was observed in a male neutered Weimaraner. Clinical signs included fecal incontinence, lethargy, moderate paraparesis, proprioceptive pelvic limb ataxia, falling, cognitive decline, incoordination, decreased interest in food, changes in posture, and episodes of trance-like behavior. Neurologic signs were first observed at approximately 4 years, 10 months of age and progressed slowly. Magnetic resonance imaging showed generalized brain atrophy with areas of white matter pathology. Humane euthanasia was elected at 6 years, 7 months of age due to increasing severity of the neurological signs. Autofluorescent intracellular granules were observed in the cerebral and cerebellar cortexes, optic nerve, and cardiac muscle of the affected dog. These abnormal inclusions in the cerebral cortex and cardiac muscle immunolabeled with antibodies to mitochondrial ATP synthase subunit c protein, like that observed in the neuronal ceroid lipofuscinosis group of lysosomal storage diseases. Immunolabeling also demonstrated pronounced neuroinflammation in brain tissues. The ultrastructural appearances of the disease-related inclusion bodies in the brain and optic nerve were quite variable. The ultrastructure and locations of many of the inclusions in the nervous tissues suggested that they were derived, at least in part, from the myelin surrounding axons. The storage bodies in the cardiac muscle were located in mitochondria-rich regions and consisted of parallel arrays of membrane-like components interspersed with electron-dense flocculent material. The disease was characterized by pronounced abnormalities in the myelin of the brain and optic nerve consisting of distinctive areas of ballooning between the layers of myelin. The whole genome sequence generated from the affected dog contained a homozygous G-to-A missense mutation in CNP, which encodes proteins with CNPase enzyme activity and a structural role in myelin. The mutation predicts a Thr42Met amino acid sequence substitution. Genotyping of archived Weimaraner DNA samples identified an additional G > A variant homozygote with a clinical history and brain lesions similar to those of the proband. Of 304 Weimaraners and over 4000 other dogs of various breeds, the proband and the other Weimaraner that exhibited similar signs were the only two that were homozygous for the CNP missense variant. CNPase immunolabeling was widespread in brain tissues from normal dogs but was undetectable in the same tissues from the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the late-onset Weimaraner disorder likely results from the missense mutation that results in CNPase deficiency, leading to myelin abnormalities, accumulation of lysosomal storage bodies, and brain atrophy. Similar disorders have been associated with different CNP variants in Dalmatians and in human subjects.

Anchor Clustering for million-scale immune repertoire sequencing data.

BACKGROUND: The clustering of immune repertoire data is challenging due to the computational cost associated with a very large number of pairwise sequence comparisons. To overcome this limitation, we developed Anchor Clustering, an unsupervised clustering method designed to identify similar sequences from millions of antigen receptor gene sequences. First, a Point Packing algorithm is used to identify a set of maximally spaced anchor sequences. Then, the genetic distance of the remaining sequences to all anchor sequences is calculated and transformed into distance vectors. Finally, distance vectors are clustered using unsupervised clustering. This process is repeated iteratively until the resulting clusters are small enough so that pairwise distance comparisons can be performed. RESULTS: Our results demonstrate that Anchor Clustering is faster than existing pairwise comparison clustering methods while providing similar clustering quality. With its flexible, memory-saving strategy, Anchor Clustering is capable of clustering millions of antigen receptor gene sequences in just a few minutes. CONCLUSIONS: This method enables the meta-analysis of immune-repertoire data from different studies and could contribute to a more comprehensive understanding of the immune repertoire data space.

An early clinical phenotype of necrotizing meningoencephalitis in the Pug reveals similarities to multiple sclerosis in humans.

Necrotizing meningoencephalitis (NME) is a fatal neuroinflammatory disease that previously carried a uniformly grave prognosis. Our recent identification of a novel early form of NME in Pugs suggests that disease onset and progression are likely more insidious than previously recognized and provides new hope that early therapeutic intervention may halt disease progression and ultimately prevent or cure NME. This novel perspective also sheds new light on the clinical similarities to multiple sclerosis (MS) in humans and provides a rationale for cross-species translation. The history of recent scientific discoveries in NME and new parallels between MS and NME will be reviewed.

Canine peripheral blood TCRαß T cell atlas: Identification of diverse subsets including CD8A+ MAIT-like cells by combined single-cell transcriptome and V(D)J repertoire analysis.

The dog is valued as a companion animal and increasingly recognized as a model for human disorders. Given the importance of T cells in health and disease, comprehensive knowledge of canine T cells can contribute to our understanding of pathogenesis mechanisms and inform the development of new treatment strategies. However, the diversity of canine T cells is still poorly understood mainly due to the lack of species-reactive antibodies for use in flow cytometry. The aim of this study was to generate a detailed atlas of peripheral blood TCRαß+ T cells of healthy dogs using single-cell RNA-sequencing (scRNAseq) combined with immune repertoire sequencing. A total of 22 TCRαß+ T cell clusters were identified, which were classified into three major groups: CD4-dominant (11 clusters), CD8A-dominant (8 clusters), and CD4/CD8A-mixed (3 clusters). Based on differential gene expression, distinct differentiation states (naïve, effector, memory, exhausted) and lineages (e.g. CD4 T helper and regulatory T cells) could be distinguished. Importantly, several T cell populations were identified, which have not been described in dogs before. Of particular note, our data provide first evidence for the existence of canine mucosa-associated invariant T cell (MAIT)-like cells, representing one of three newly identified FCER1G+ innate-like CD8A+ T cell populations in the peripheral blood of healthy dogs. In conclusion, using scRNAseq combined with immune repertoire sequencing we were able to resolve canine TCRαß+ T cell populations at unprecedented resolution. The peripheral blood TCRαß+ T cell atlas of healthy dogs generated here represents an important reference data set for future studies and is of relevance for identifying new targets for T cell-specific therapies.

B-1 plasma cells require non-cognate CD4 T cell help to generate a unique repertoire of natural IgM.

Evolutionarily conserved, "natural" (n)IgM is broadly reactive to both self and foreign antigens. Its selective deficiency leads to increases in autoimmune diseases and infections. In mice, nIgM is secreted independent of microbial exposure to bone marrow (BM) and spleen B-1 cell-derived plasma cells (B-1PC), generating the majority of nIgM, or by B-1 cells that remain non-terminally differentiated (B-1sec). Thus, it has been assumed that the nIgM repertoire is broadly reflective of the repertoire of body cavity B-1 cells. Studies here reveal, however, that B-1PC generate a distinct, oligoclonal nIgM repertoire, characterized by short CDR3 variable immunoglobulin heavy chain regions, 7-8 amino acids in length, some public, many arising from convergent rearrangements, while specificities previously associated with nIgM were generated by a population of IgM-secreting B-1 (B-1sec). BM, but not spleen B-1PC, or B-1sec also required the presence of TCRαß CD4 T cells for their development from fetal precursors. Together, the studies identify important previously unknown characteristics of the nIgM pool.

Read at home to do well at school: informal reading predicts achievement and motivation in English as a foreign language.

Introduction: Learning English as a foreign language is necessary for many students to actively participate in an increasingly globalized world. This study explores the role of informal English language engagement for students' reading and listening skills, as well as motivation to learn English. In an era of global interconnectedness, informal learning gains importance as a supplement to formal education. Methods: This study extends the evidence base by analyzing extramural reading and listening activities in a large-scale longitudinal investigation involving secondary school learners (N = 1,994) in Germany. Results: Our results show that frequent informal reading significantly relates to increases in students' English comprehension skills and their motivation for language learning, reaffirming previous cross-sectional findings. Discussion: The results highlight the relevance of informal language activities for effective language learning and students' English as a foreign language motivation. Additionally, discrepancies between reading and listening outcomes are discussed.

Seroprevalence of SARS-CoV-2 in 1186 Equids Presented to a Veterinary Medical Teaching Hospital in California from 2020 to 2022.

While some companion animals have been shown to be susceptible to SARS-CoV-2, their role in the COVID-19 pandemic has remained poorly investigated. Equids are susceptible to SARS-CoV-2 based on the similarity of the human ACE-2 receptor and reports of infection. Clinical disease and prevalence factors associated with SARS-CoV-2 infection in equids have not yet been investigated. The aim of this study was to determine the seroprevalence of SARS-CoV-2 and selected prevalence factors in 1186 equids presented for various conditions to a Veterinary Medical Teaching Hospital over a two-year period. Blood samples were tested for SARS-CoV-2 antibodies using an ELISA targeting the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Further, selected prevalence factors (season, age, breed, sex, presenting complaint) were retrieved from the medical records. No information was available on whether the horses had come into contact with COVID-19-positive individuals. Among the study animals, 42/1186 (3.5%) horses had detectable SARS-CoV-2 antibodies. Amongst the prevalence factors investigated, only seasonality (spring) was associated with a greater frequency of seropositivity to SARS-CoV-2. Horses with medical and surgical complaints were more likely to test seropositive to SARS-CoV-2 compared to horses presented for routine health care procedures, suggesting more frequent and/or longer interactions with individuals with COVID-19. While horses can become infected with SARS-CoV-2 via the occasional spillover from COVID-19 individuals, clinical disease expression remains subclinical, making horses an unlikely contributor to the spread of SARS-CoV-2.

Evaluation of clonality from multiple anatomic sites in canine epitheliotropic T cell lymphoma.

BACKGROUND: Canine epitheliotropic cutaneous T-cell lymphoma (eCTCL) is thought to represent a disease homologue to human mycosis fungoides (MF). In human MF, neoplastic cells are phenotypically consistent with resident effector memory T cells, a population that remains for an extended period within tissue without circulating. Dogs with eCTCL often present with lesions in multiple locations, raising the question of whether the neoplasm is of the same T-cell subpopulation or not. OBJECTIVES: To characterize the antigen receptor gene rearrangements of lymphocytes from skin and blood of dogs with eCTCL to determine if neoplastic clones are identical. ANIMALS: Fourteen dogs with eCTCL. MATERIALS AND METHODS: Histological and immunohistochemical examination, and PCR for antigen receptor rearrangement (PARR) for T-cell receptor gamma (TRG) performed on multiple cutaneous biopsy samples and blood. RESULTS: All skin biopsies contained cluster of differentiation (CD)3-positive neoplastic lymphocytes. Within individual dogs, all skin biopsies revealed identical TRG clonality profiles, suggesting that the same neoplastic clone was present in all sites. In the blood, a matching clone was found in six of 14 dogs, a unique clone was observed in nine of 14 dogs, and no clone was detected in two of 14 dogs. CONCLUSIONS: These findings show that canine eCTCL lesions in multiple locations harbour the same neoplastic clone, neoplastic lymphocytes do not remain fixed to the skin and instead can circulate via blood, differing clones can be identified in skin versus blood, and circulating neoplastic cells can be detected without lymphocytosis.

Contexte - On pense que le lymphome T cutané épithéliotrope canin (eCTCL) représente une maladie homologue au mycosis fongoïde (MF) humain. Dans le MF humain, les cellules néoplasiques sont phénotypiquement compatibles avec les cellules T mémoire effectrices résidentes, une population qui reste pendant une période prolongée dans les tissus sans circuler. Les chiens atteints d'eCTCL présentent souvent des lésions à plusieurs endroits, ce qui soulève la question de savoir si le néoplasme appartient ou non à la même sous-population de lymphocytes T. Objectifs - Caractériser les réarrangements du gène du récepteur antigénique des lymphocytes de la peau et du sang des chiens atteints d'eCTCL afin de déterminer si les clones néoplasiques sont identiques. Animaux - Quatorze chiens avec eCTCL. Matériels et méthodes - Examen histologique et immunohistochimique, et PCR pour le réarrangement des récepteurs antigéniques (PARR) pour le récepteur gamma des lymphocytes T (TRG) effectués sur plusieurs échantillons de biopsie cutanée et de sang. Résultats - Toutes les biopsies cutanées contenaient des amas de lymphocytes néoplasiques positifs à la différenciation (CD)3. Chez les chiens individuels, toutes les biopsies cutanées ont révélé des profils de clonalité TRG identiques, suggérant que le même clone néoplasique était présent dans tous les sites. Dans le sang, un clone correspondant a été trouvé chez six des 14 chiens, un clone unique a été observé chez neuf des 14 chiens et aucun clone n'a été détecté chez deux des 14 chiens. Conclusions - Ces résultats montrent que les lésions eCTCL canines à plusieurs endroits abritent le même clone néoplasique, les lymphocytes néoplasiques ne restent pas fixés à la peau et peuvent plutôt circuler par le sang, différents clones peuvent être identifiés dans la peau par rapport au sang, et les cellules néoplasiques circulantes peuvent être détecté sans lymphocytose.

Introducción- se cree que el linfoma epiteliotrópico cutáneo de células T canino (eCTCL) representa una enfermedad homóloga a la micosis fungoide (MF) humana. En la MF humana, las células neoplásicas son fenotípicamente consistentes con las células T de memoria efectoras residentes, una población que permanece durante un período prolongado dentro del tejido sin circular. Los perros con eCTCL a menudo presentan lesiones en múltiples ubicaciones, lo que plantea la cuestión de si la neoplasia es de la misma subpoblación de células T o no. Objetivos- caracterizar los reordenamientos del gen del receptor de antígeno de los linfocitos de la piel y la sangre de perros con eCTCL para determinar si los clones neoplásicos son idénticos. Animales- catorce perros con eCTCL. Materiales y métodos - Examen histológico e inmunohistoquímico, y PCR para el reordenamiento del receptor de antígeno (PARR) para el receptor de células T gamma (TRG) realizado en múltiples muestras de biopsia cutánea y sangre. Resultados- todas las biopsias de piel contenían linfocitos neoplásicos positivos para grupos de diferenciación (CD)3. Dentro de perros individuales, todas las biopsias de piel revelaron perfiles de clonalidad de TRG idénticos, lo que sugiere que el mismo clon neoplásico estaba presente en todos los sitios. En la sangre, se encontró un clon compatible en seis de 14 perros, se observó un clon único en nueve de 14 perros y no se detectó ningún clon en dos de 14 perros. Conclusiones- estos hallazgos muestran que las lesiones de eCTCL canino en múltiples ubicaciones albergan el mismo clon neoplásico, los linfocitos neoplásicos no permanecen fijados a la piel y, en cambio, pueden circular a través de la sangre, se pueden identificar diferentes clones en la piel versus la sangre y las células neoplásicas circulantes pueden ser identificadas sin presencia de linfocitosis.

Contexto - Acredita-se que o linfoma epiteliotrópico cutâneo de células T canino (eCTCL) representa uma doença análoga à micose fungoide (MF) humana. Na MF humana, as células neoplásicas são fenotipicamente consistentes com células T efetoras de memória residentes, uma população que permanece por um período extenso no tecido sem entrar na circulação. Os cães com eCTCL frequentemente apresentam lesões em múltiplos locais, levantando a questão de se a neoplasia é da mesma subpopulação de células T ou não. Objetivos - Caracterizar os rearranjos dos genes receptores de antígenos dos linfócitos da pele e do sangue de cães com eCTCL para determinar se os clones neoplásicos são idênticos. Animais - Quatorze cães com eCTCL. Materiais e métodos - Exame histológico e imunohistoquímico, e PCR para rearranjo de receptor de antígeno (PARR) para o receptor Gama de células T (TRG) realizado em múltiplas amostras de biópsia cutânea e sangue. Resultados - Todas as biópsias cutâneas continham clusters de diferenciação linfócitos T (CD)3- positivos. Entre os indivíduos, todas as biópsias cutâneas revelaram perfis de clonalidade de TGR idênticos em seis dos 14 cães, sugerindo que a mesma célula neoplásica estava presente em todos os locais. No sangue, um clone correspondente foi encontrado em seis dos 14 cães, um clone único foi observado em nove dos 14 cães e nenhum clone foi detectado em dois dos 14 cães. Conclusões - Estes achados demonstraram que as lesões de eCTCL em múltiplos locais possuem o mesmo clone neoplásico, linfócitos neoplásicos não permanecem fixos na pele e podem circular por via sistêmica , diversos tipos de clones podem ser identificados na pele versus sangue, e as células neoplásicas circulantes podem ser detectadas sem linfocitose.

A potential early clinical phenotype of necrotizing meningoencephalitis in genetically at-risk pug dogs.

BACKGROUND: Necrotizing meningoencephalitis (NME) in the pug dogs is a fatal neuroinflammatory disease associated with rapid progression and poor response to conventional immunosuppressive therapy. Diagnosis is typically made after severe neurological abnormalities have manifested. HYPOTHESIS/OBJECTIVE: Pug dogs at genetic risk for NME might manifest neurological abnormalities before developing pathognomonic clinical signs of NME. ANIMALS: Thirty-six pug dogs less than 4 years of age asymptomatic for NME. METHODS: Prospective observational cohort study with germline genome-wide genotyping. Neurological examinations were performed 4 weeks apart to document reproducible findings of central nervous system disease. Magnetic resonance imaging, cerebrospinal fluid analysis, and testing for infectious diseases were performed in all pugs with reproducible abnormalities detected on neurological examination. RESULTS: The overall risk allele frequency in this cohort was 40%; 5 (14%) dogs were high risk, 19 (53%) dogs were medium risk, and 12 (33%) dogs were low genetic risk for NME. Reproducible abnormalities detected on neurological examination were identified in 8/24 (33%) genetically at-risk dogs and 0/12 (0%) low risk dogs. Clinical abnormalities included multifocal spinal pain in 8/8, reduced menace response in 5/8, and lateralizing postural reaction deficits in 5/8 pugs. There was a strong association between genotype risk and the presence of this clinical phenotype (P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Our findings suggest the presence of a novel early clinical phenotype of NME in apparently asymptomatic genetically at-risk pugs which might be used to plan early diagnostic and therapeutic clinical trials.

Novel clonality assays for T cell lymphoma in cats targeting the T cell receptor beta, T cell receptor delta, and T cell receptor gamma loci.

BACKGROUND: T cell clonality assays in veterinary medicine currently target only the T cell receptor gamma (TRG) locus. Existing assays have suboptimal sensitivity because of insufficient primer coverage of all possible rearrangements. OBJECTIVE: Develop higher sensitivity clonality assays targeting the TRG, delta (TRD), and beta (TRB) loci in cats. ANIMALS: Cats with histopathologically confirmed lymphoma (n = 89), non-lymphoma (n = 35), and possible hepatic small cell lymphoma (n = 31). METHODS: Molecular clonality assay development utilizing our recently reported topology and expressed repertoire data of the T cell receptor loci in cats. Determination of clonality status of lymphoma, non lymphoma, and possible hepatic small cell lymphoma samples, and calculation of assay sensitivity and specificity. RESULTS: The new multiplex TRG assay yielded the highest sensitivity (95.5%). All assays yielded 100% specificity except for the new multiplex TRG assay (97.3%). The combination of the new TRG and TRB assays yielded sensitivity of 98.9% and specificity of 97.0%. The new TRG assay detected clonality in 17/31 possible small cell lymphoma livers, whereas an existing TRG assay detected clonality in 6/31 livers. CONCLUSIONS AND CLINICAL IMPORTANCE: The assessment of multiple T cell loci compensates for the potential shortcomings of individual assays. Using a combination of molecular clonality assays will increase the overall sensitivity for the diagnosis of T-cell lymphoma in cats, especially intestinal, and hepatic small cell lymphoma. Hepatic small cell lymphomas detected by the new TRG assay utilized rarely expressed V and J genes not recognized by previous assays, likely indicating unique biology of hepatic small cell lymphoma in cats.

Leukocyte and cytokine variables in asymptomatic Pugs at genetic risk of necrotizing meningoencephalitis.

BACKGROUND: Necrotizing meningoencephalitis (NME, aka Pug dog encephalitis) is an inflammatory brain condition associated with advanced disease at initial presentation, rapid progression, and poor response to conventional immunomodulatory therapy. HYPOTHESIS/OBJECTIVES: That genetic risk for NME, defined by a common germline DNA haplotype located on chromosome 12, is associated with altered blood cytokine concentrations and leukocyte subsets in asymptomatic Pugs. ANIMALS: Forty Pug dogs asymptomatic for NME from a hospital sample. METHODS: Prospective observational cohort study, including germline genome-wide genotyping, plasma cytokine determination by multiplexed profiling, and leukocyte subset characterization by flow cytometric analysis. RESULTS: Seven (18%) dogs were high risk, 10 (25%) medium risk, and 23 (58%) low risk for NME, giving a risk haplotype frequency of 30%. High and medium risk Pugs had significantly lower proportion of CD4+ T cells (median 22% [range, 7.3%-38%] vs 29% [range, 16%-41%], P = .03) and higher plasma IL-10 concentrations than low-risk Pugs (median 14.11 pg/mL [range, 9.66-344.19 pg/mL] vs 12.21 pg/mL [range, 2.59-18.53 pg/mL], P = .001). No other variables were significantly associated with the NME haplotype-based risk. CONCLUSIONS AND CLINICAL IMPORTANCE: These data suggest an immunological underpinning to NME and a biologic rationale for future clinical trials that investigate novel diagnostic, preventative, and therapeutic strategies for this disease.

Use of immune repertoire sequencing to resolve discordant microscopic and immunochemical findings in a case of T cell-rich large B cell lymphoma in a young dog.

BACKGROUND: Lymphocytic neoplasms with frequent reactive lymphocytes are uncommonly reported in dogs, and can pose a diagnostic challenge. Different diagnostic modalities such as cytology, flow cytometry, histopathology, immunohistochemistry, and clonality testing, are sometimes required for a diagnosis. This report illustrates the value of using a multi-modal diagnostic approach to decipher a complex lymphocytic tumor, and introduces immune repertoire sequencing as a diagnostic adjunct. CASE PRESENTATION: A 10-month-old Great Dane was referred for marked ascites. Cytologic analysis of abdominal fluid and hepatic aspirates revealed a mixed lymphocyte population including numerous large lymphocytes, yielding a diagnosis of lymphoma. Flow cytometrically, abdominal fluid lymphocytes were highly positive for CD4, CD5, CD18, CD45, and MHC II, consistent with T cell lymphoma. Due to a rapidly deteriorating clinical condition, the dog was euthanized. Post mortem histologic evaluation showed effacement of the liver by aggregates of B cells surrounded by T cells, suggestive of hepatic T cell-rich large B cell lymphoma. Immune repertoire sequencing confirmed the presence of clonal B cells in the liver but not the abdominal fluid, whereas reactive T cells with shared, polyclonal immune repertoires were found in both locations. CONCLUSIONS: T cell-rich large B cell lymphoma is a rare neoplasm in dogs that may be challenging to diagnose and classify due to mixed lymphocyte populations. In this case, the results of histopathology, immunohistochemistry and immune repertoire sequencing were most consistent with a hepatic B cell neoplasm and reactive T cells exfoliating into the abdominal fluid. Immune repertoire sequencing was helpful in delineating neoplastic from reactive lymphocytes and characterizing repertoire overlap in both compartments. The potential pitfalls of equating atypical cytomorphology and monotypic marker expression in neoplasia are highlighted.

Is a Long Essay Always a Good Essay? The Effect of Text Length on Writing Assessment.

The assessment of text quality is a transdisciplinary issue concerning the research areas of educational assessment, language technology, and classroom instruction. Text length has been found to strongly influence human judgment of text quality. The question of whether text length is a construct-relevant aspect of writing competence or a source of judgment bias has been discussed controversially. This paper used both a correlational and an experimental approach to investigate this question. Secondary analyses were performed on a large-scale dataset with highly trained raters, showing an effect of text length beyond language proficiency. Furthermore, an experimental study found that pre-service teachers tended to undervalue text length when compared to professional ratings. The findings are discussed with respect to the role of training and context in writing assessment.

Using pre-prandial blood glucose to assess eating in the absence of hunger in free-living individuals.

Our ability to understand and intervene on eating in the absence of hunger (EAH) as it occurs in peoples' natural environments is hindered by biased methods that lack ecological validity. One promising indicator of EAH that does not rely on self-report and is easily assessed in free-living individuals is glucose. Here, we hypothesize that elevated pre-prandial blood glucose concentrations (PPBG), which reflect a source of readily-available, short-term energy, are a biological indicator of EAH. This was a 7-day observational study of N = 41, 18-24 year old men and women with BMI < 25 kg/m2 (60%) or BMI ≥ 25 kg/m2 (40%). We collected data using ecological momentary assessment from people in their natural environments. We defined EAH by self-report (perceived EAH) and by PPBG thresholds using two methods (standardized, PPBG < 85 mg/dl; personalized, PPBG < individual fasting levels). Multilevel modeling was used to analyze the data. N = 963 eating events were reported. There were significantly (p < .05) fewer perceived EAH events (25%) as compared to standardized (62%) and personalized PPBG-defined EAH events (51%). Consistent with published literature, perceived EAH was more likely to occur at a higher PPBG (p < .01), particularly among participants with a BMI ≥ 25 kg/m2 (pint < .01). Additionally, discordance between perceived EAH and PPBG-defined EAH, indicating a perception of hunger at an eating event when PPBS was elevated, was less likely among participants with a BMI < 25 kg/m2 vs. those with a BMI ≥ 25 kg/m2 (pint < .01) as well as at snacks vs. meals (pint < .01). These findings provide preliminary support for using PPBG as a biological indicator of EAH in free-living individuals.

Assessment of 10-nm Particle Number (PN) Portable Emissions Measurement Systems (PEMS) for Future Regulations.

The particle number (PN) emissions of vehicles equipped with particulate filters are low. However, there are technologies that can have high PN levels, especially below the currently lower regulated particle size of 23 nm. Sub-23-nm particles are also considered at least as dangerous as the larger ultrafine particles. For this reason, the European Union (EU) is planning to regulate particles down to 10 nm. In this study we compared prototype portable emission measurement systems (PEMS) and reference laboratory systems measuring from 10 nm. The tests included cycles and constant speeds, using vehicles fuelled with diesel, gasoline or liquefied petroleum gas (LPG). The results showed that the PEMS were within ±40% of the reference systems connected to the tailpipe and the dilution tunnel. Based on the positive findings and the detection efficiencies of the prototype instruments, a proposal for the technical specifications for the future regulation was drafted.

Topology and expressed repertoire of the Felis catus T cell receptor loci.

BACKGROUND: The domestic cat (Felis catus) is an important companion animal and is used as a large animal model for human disease. However, the comprehensive study of adaptive immunity in this species is hampered by the lack of data on lymphocyte antigen receptor genes and usage. The objectives of this study were to annotate the feline T cell receptor (TR) loci and to characterize the expressed repertoire in lymphoid organs of normal cats using high-throughput sequencing. RESULTS: The Felis catus TRG locus contains 30 genes: 12 TRGV, 12 TRGJ and 6 TRGC, the TRB locus contains 48 genes: 33 TRBV, 2 TRBD, 11 TRBJ, 2 TRBC, the TRD locus contains 19 genes: 11 TRDV, 2 TRDD, 5 TRDJ, 1 TRDC, and the TRA locus contains 127 genes: 62 TRAV, 64 TRAJ, 1 TRAC. Functional feline V genes form monophyletic clades with their orthologs, and clustering of multimember subgroups frequently occurs in V genes located at the 5' end of TR loci. Recombination signal (RS) sequences of the heptamer and nonamer of functional V and J genes are highly conserved. Analysis of the TRG expressed repertoire showed preferential intra-cassette over inter-cassette rearrangements and dominant usage of the TRGV2-1 and TRGJ1-2 genes. The usage of TRBV genes showed minor bias but TRBJ genes of the second J-C-cluster were more commonly rearranged than TRBJ genes of the first cluster. The TRA/TRD V genes almost exclusively rearranged to J genes within their locus. The TRAV/TRAJ gene usage was relatively balanced while the TRD repertoire was dominated by TRDJ3. CONCLUSIONS: This is the first description of all TR loci in the cat. The genomic organization of feline TR loci was similar to that of previously described jawed vertebrates (gnathostomata) and is compatible with the birth-and-death model of evolution. The large-scale characterization of feline TR genes provides comprehensive baseline data on immune repertoires in healthy cats and will facilitate the development of improved reagents for the diagnosis of lymphoproliferative diseases in cats. In addition, these data might benefit studies using cats as a large animal model for human disease.

MicroRNA profiling in canine multicentric lymphoma.

Lymphoma is the most common hematopoietic tumour in dogs and is remarkably similar to the human disease. Tumour biomarker discovery is providing new tools for diagnostics and predicting therapeutic response and clinical outcome. MicroRNAs are small non-coding RNAs that participate in post-transcriptional gene regulation and their aberrant expression can impact genes involved in cancer. The aim of this study was to characterize microRNA expression in lymph nodes and plasma from dogs with multicentric B or T cell lymphoma compared to healthy control dogs. We further compared expression between lymph nodes and corresponding plasma samples and assessed changes in expression at relapse compared to time of diagnosis. Lastly, we investigated microRNAs for association with clinical outcome in patients treated with CHOP chemotherapy. A customized PCR array was utilized to profile 38 canine target microRNAs. Quantification was performed using real time RT-qPCR and relative expression was determined by the delta-delta Ct method. In lymph nodes, there were 16 microRNAs with significantly altered expression for B cell lymphoma and 9 for T cell lymphoma. In plasma, there were 15 microRNAs altered for B cell lymphoma and 3 for T cell lymphoma. The majority of microRNAs did not have correlated expression between lymph node and plasma and only 8 microRNAs were significantly different between diagnosis and relapse. For B cell lymphoma, 8 microRNAs had differential expression in the non-remission group compared to dogs that completed CHOP in complete remission. Four of these microRNAs were also altered in patients that died prior to one-year. Kaplan-Meier survival curves for high versus low microRNA expression revealed that 10 microRNAs were correlated with progression-free survival and 3 with overall survival. This study highlights microRNAs of interest for canine multicentric lymphoma. Future goals include development of microRNA panels that may be useful as biomarkers with the intent to provide improved outcome prediction to veterinary cancer patients.

Composite lymphoma of concurrent T zone lymphoma and large cell B cell lymphoma in a dog.

BACKGROUND: Evolution of indolent to aggressive lymphoma has been described in dogs but is difficult to distinguish from the de novo development of a second, clonally distinct lymphoma. Differentiation of these scenarios can be aided by next generation sequencing (NGS)-based assessment of clonality of lymphocyte antigen receptor genes. CASE PRESENTATION: An 8-year-old male intact Mastiff presented with generalized lymphadenomegaly was diagnosed with nodal T zone lymphoma (TZL) based on cytology, histopathology, immunohistochemistry and flow cytometry. Thirteen months later, the dog re-presented with progressive lymphadenomegaly, and based on cytology and flow cytometry, a large B cell lymphoma (LBCL) was diagnosed. Sequencing-based clonality testing confirmed the de novo development of a LBCL and the persistence of a TZL. CONCLUSIONS: The occurrence of two distinct lymphoid neoplasms should be considered if patient features and tumor cytomorphology or immunophenotype differ among sequential samples. Sequencing-based clonality testing may provide conclusive evidence of two concurrent and distinct clonal lymphocyte populations, termed most appropriately "composite lymphoma".

A review of canine B cell clonality assays and primer set optimization using large-scale repertoire data.

Several molecular clonality assays have been developed to assess canine B cell proliferations. These assays were based on different sequence data, utilized different assay designs and employed different testing strategies. This has resulted in a complex body of literature and complicates evidence-based selection of primer sets. In addition, further refinement of primer sets is difficult because it is unknown how well current primer sets cover the expressed sequence repertoire. The objectives of this study were 1) to provide an overview of published IGH clonality assays that highlights key differences in assay design and testing strategy and 2) to propose a novel method for optimizing primer sets that leverages large-scale sequencing data. A review of previously published assays highlighted confounding factors that hamper a direct comparison of performance metrics between studies. These findings illustrate the need for a multi-institutional effort to harmonize veterinary clonality testing. A novel in silico analysis of primer sequences using a large dataset of expressed sequences identified shortfalls of existing primer sets and was used to guide primer optimization. Three optimized primer sets were tested and yielded qualitative sensitivity values between 80-90%. The qualitative sensitivity ranged from 1% to over 50% and was dependent on the size of the neoplastic clone and the sample DNA used. These findings illustrate that inclusion of high-throughput sequencing data for primer design can be a useful tool to guide primer design and optimization. This strategy could be applied to other antigen receptor loci or species to further improve veterinary clonality assays.