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The impact of different acquisition times (AqT) and technetium-99 m-labeled (

Supplemental Movie S2. Follicular motility of B cells. The Movie complements Figure 4J.

Supplementary Movie S1. Follicular motility of Emu-Tcl1 leukemia cells. The Movie complements Figure 4J.

Figure S1. Gating strategy to characterize and quantitate CD19+CD5+ leukemia Emu-Tcl1 cell frequencies and homeostatic chemokine receptor expression on splenic, LN, PB, and BM derived CD19+CD5+ gated Emu-Tcl1 tumor cells. This Figure complements Figure 1. Figure S2. Surface marker expression of murine Emu-Tcl1 leukemia cells is shown. Human primary B-CLL cells share characteristic expression of CXCR5. This Figure complements Figure 1. Figure S3. Enrichment plots of seven proliferation-related signatures are presented. Short term transfer of CXCR5+/+Emu-Tcl1 or CXCR5-/-Emu-Tcl1 tumor cells shows upregulation of the gene encoding the cell-cycle inhibitor p21 in CXCR5-deficient leukemia cells. Growth and activation delay of CXCR5-/- Emu-Tcl1 tumor cells can be reversed by direct contacts with stromal cells in vitro. This Figure complements Figure 2. Figure S4. (A) shows that CCR7-deficiency on leukemia cells does not affect their migratory behavior and complements Figure 3. (B) Ebi2-/-Emu-Tcl1 leukemia cells enter B cell follicles and the GC light zone area in a pattern comparable to Ebi2++Emu-Tcl1 leukemia cells, as presented in Figure 4, and (C) tumor growth of Ebi2-/-Emu-Tcl1 mice is unaltered compared to Wt Emu-Tcl1 mice. Figure S5. Human primary B-CLLs exhibit a characteristic FDC staining. This Figure complements Figure 4. Figure S6. Pertussis toxin (PTX) treatment induces apoptosis in Emu-Tcl leukemia cells co-cultured with FDC-HK stroma cells. This Figure complements Figure 4. Figure S7. Intravital application of anti-CD21/CD35 Fab fragments stains specifically GC light zone localized FDCs. These stainings complement Figure 5. Figure S8. Challenge of Rag2-/- mice with Emu-Tcl1 leukemia B cells induces FDC networks and a tumor cell-promoting cytokine and chemokine secretion. This Figure accompanies Figure 6. Figure S9. Abrogation of LTbetaR-signaling leads to disappearance of characteristic stromal elements within the B cell follicle; however, CD31+ blood vessels are not affected. In accordance with Figure 7, Ltalpha -/- Emu-Tcl1 mice exhibit substantially delayed leukemia growth and progression.

Lack of hypoxia and tip cell signature

Neutrophil localization in human SMZL and benign spleen tissues.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Tumor-induced down- and upregulations of genes in the stroma.

Top GO and GSEA pathways

Tumor-induced remodeling of the microenvironment relies on the formation of blood vessels, which go beyond the regulation of metabolism, shaping a maladapted survival niche for tumor cells. In high-grade B-cell lymphoma, angiogenesis correlates with poor prognosis, but attempts to target established proangiogenic pathways within the vascular niche have been inefficient. Here, we analyzed Myc-driven B-cell lymphoma–induced angiogenesis in mice. A few lymphoma cells were sufficient to activate the angiogenic switch in lymph nodes. A unique morphology of dense microvessels emerged without obvious tip cell guidance and reliance on blood endothelial cell (BEC) proliferation. The transcriptional response of BECs was inflammation independent. Conventional HIF1α or Notch signaling routes prevalent in solid tumors were not activated. Instead, a nonconventional hypersprouting morphology was orchestrated by lymphoma-provided VEGFC and lymphotoxin (LT). Interference with VEGF receptor-3 and LTβ receptor signaling pathways abrogated lymphoma angiogenesis, thus revealing targets to block lymphomagenesis.Significance:In lymphoma, transcriptomes and morphogenic patterns of the vasculature are distinct from processes in inflammation and solid tumors. Instead, LTβR and VEGFR3 signaling gain leading roles and are targets for lymphomagenesis blockade.

Legends for Supplementary Figures S1-S6.

Supplementary Material+Methods, Legends, References

Recruitment of tumor-associated macrophages and neutrophils (TAM and TAN) to solid tumors contributes to immunosuppression in the tumor microenvironment; however, their contributions to lymphoid neoplasms are less clear. In human chronic lymphocytic leukemia (CLL), tumor B cells lodge in lymph nodes where interactions with the microenvironment occur. Tumor cell homing stimulates proliferation, such that engagement of the B-cell receptor is important for malignant progression. In the Eμ-Tcl1 murine model of CLL, we identified gene expression signatures indicative of a skewed polarization in the phenotype of monocytes and neutrophils. Selective ablation of either of these cell populations in mice delayed leukemia growth. Despite tumor infiltration of these immune cells, a systemic inflammation was not detected. Notably, in progressive CLL, splenic neutrophils were observed to differentiate toward a B-cell helper phenotype, a process promoted by the induction of leukemia-associated IL10 and TGFβ. Our results suggest that targeting aberrant neutrophil differentiation and restoring myeloid cell homeostasis could limit the formation of survival niches for CLL cells. Cancer Res; 76(18); 5253–65. ©2016 AACR.

To the authorsʼ knowledge there is only one report in the literature, dealing with paramedullary plasmacytomas in horses as so-called “break out lesions”, originating from the bone marrow of a vertebra and invading the epidural space (Drew and Greatorex 1974). Our case report describes the clinical and patho-morphological findings of such a case. The affected 8-month-old warmblood weanling colt exhibited acute tetraparesis thus, a spinal cord lesion in the cervical column was suspected. Computed tomography revealed osteolytic lesions in C1 and a heterogeneous mass within the respective segment of the vertebral canal. By means of patho-morphological examination, with the aid of transmission electron microscopy, a plasmacytoma was identified, that had grown out of the bone marrow of C1 into the epidural space, locally compressing the spinal cord. In tetraparetic, young horses traumatic spine cord injuries, cervical vertebral malformations and infectious diseases of the central nervous system represent common differentials. However bone-dissolving tumours such as paramedullary plasmacytomas should be considered as a further possible cause in the presence of severe neurological deficits.

Insight into processes that determine CD8(+) T cell memory formation has been obtained from infection models. These models are biased toward an inflammatory milieu and often employ high avidity CD8(+) T cells in adoptive transfer procedures. It is unclear whether these conditions mimic the differentiation processes of an endogenous repertoire that proceed upon non-inflammatory conditions prevailing in premalignant tumor lesions. We examined the role of cytolytic capacity on CD8(+) T cell fate decisions when primed by tumor cells or by minor histocompatibility antigen-mismatched leukocytes. CD8(+) memory commitment was analyzed in Ebag9-deficient mice that exhibit an enhanced tumor cell lysis. This property endowed Ebag9(-/-) mice with extended control of Tcl-1 oncogene-induced chronic lymphocytic leukemia progression. In Ebag9(-/-) mice, an expanded memory population was obtained for anti-HY and anti-SV40 T antigen-specific T cells, despite unchanged effector frequencies in the primary response. By comparing the single-cell transcriptomes of CD8(+) T cells responding to tumor cell vaccination, we found differential distribution of subpopulations between Ebag9(+/+) and Ebag9(-/-) T cells. In Ebag9(-/-) cells, these larger clusters contained genes encoding transcription factors regulating memory cell differentiation, along with anti-apoptotic gene functions. Our findings link EBAG9-controlled cytolytic activity and the commitment to the CD8(+) memory lineage.

The superficial digital flexor tendon (SDFT) is the most frequently injured structure of the musculoskeletal system in sport horses and a common cause for early retirement. This project’s aim was to visualize and measure the strain of the sound, injured, and healing SDFTs in a pony during walk and trot. For this purpose, biplanar high-speed fluoroscopic kinematography (FluoKin), as a high precision X-ray movement analysis tool, was used for the first time in vivo with equine tendons. The strain in the metacarpal region of the sound SDFT was 2.86% during walk and 6.78% during trot. When injured, the strain increased to 3.38% during walk and decreased to 5.96% during trot. The baseline strain in the mid-metacarpal region was 3.13% during walk and 6.06% during trot and, when injured, decreased to 2.98% and increased to 7.61%, respectively. Following tendon injury, the mid-metacarpal region contributed less to the overall strain during walk but showed increased contribution during trot. Using this marker-based FluoKin technique, direct, high-precision, and long-term strain measurements in the same individual are possible. We conclude that FluoKin is a powerful tool for gaining deeper insight into equine tendon biomechanics.

Equine paranasal sinuses are susceptible to inflammation. Insufficient drainage through the nasal passages and meatus may lead to the accumulation of inspissated purulent discharge. Particularly in ponies, these anatomical structures are suspected to be relatively small. To date, there are no reports considering the morphology of nasal conchal bullae in small horse breeds such as Shetland ponies. The aim of the present study was to evaluate the size of the conchal bullae and the medial nasal conchae of Shetland ponies and their relation to the skull dimension using computed tomography. Reconstructed images of healthy adult heads of Shetland ponies were used. Linear skull measurements as well as two cranial indices of the head dimensions were taken. Length, width and height of the dorsal and ventral conchal bullae and the medial nasal conchae were measured in relation to the skull and compared with the data of skulls of large breed horses. The anatomical proportions of pony heads were characterized by a smaller cranial index and a greater nasal index than those of large breed horses. Shetland ponies showed a longer cranial length compared with the nasal length. Heads are consistently smaller, and the relationship of the bullae to the head length was also smaller than those measured in large breed horses. A negative correlation between the head and bullae size was found. In conclusion, this study suggests that Shetland ponies have distinguishing proportions of the head. These findings are relevant for clinical examination and surgical treatment of equine sinus disease in those breeds.

Tumor-induced remodeling of the microenvironment relies on the formation of blood vessels, which go beyond the regulation of metabolism, shaping a maladapted survival niche for tumor cells. In high-grade B-cell lymphoma, angiogenesis correlates with poor prognosis, but attempts to target established proangiogenic pathways within the vascular niche have been inefficient. Here, we analyzed Myc-driven B-cell lymphoma–induced angiogenesis in mice. A few lymphoma cells were sufficient to activate the angiogenic switch in lymph nodes. A unique morphology of dense microvessels emerged without obvious tip cell guidance and reliance on blood endothelial cell (BEC) proliferation. The transcriptional response of BECs was inflammation independent. Conventional HIF1α or Notch signaling routes prevalent in solid tumors were not activated. Instead, a nonconventional hypersprouting morphology was orchestrated by lymphoma-provided VEGFC and lymphotoxin (LT). Interference with VEGF receptor-3 and LTβ receptor signaling pathways abrogated lymphoma angiogenesis, thus revealing targets to block lymphomagenesis. Significance: In lymphoma, transcriptomes and morphogenic patterns of the vasculature are distinct from processes in inflammation and solid tumors. Instead, LTβR and VEGFR3 signaling gain leading roles and are targets for lymphomagenesis blockade.

Background Signal intensity (SI) of equine tendinopathies in MRI differs between the superficial digital flexor tendon (SDFT) and the deep digital flexor tendon (DDFT). In DDFT lesions, short tau inversion recovery (STIR) SI decreases earlier than T2-weighted (T2w) SI, while the latter decreases earlier in SDFT lesions, but long-term results using STIR sequences are lacking. Methods Standing MRI of eight horses with naturally occurring SDFT lesions was performed at the day of treatment as well as 2, 6 and 12 months after treatment. Results After 12 months, six horses were sound and showed complete resolution of increased SI in T2w fast spin echo (FSE) images, but increased SI was found in STIR images in three horses and persisted in T1w and T2*w gradient recall echo images of all horses. In contrast, hyperintense areas were still visible in the SDFT in T2w FSE images in two horses presenting with re-injury. In the six horses without re-injury, percentage of cross-sectional areas affected and SI decreased over time in all sequences. Conclusions This study suggests that SI in naturally occurring SDFT lesions decreases earlier in T2w FSE than in STIR images, in contrast to the DDFT.

In magnetic resonance imaging (MRI) examinations, moderate to severe changes of the distal sesamoidean impar ligament (DSIL) were found in horses with lameness localized to their feet. Histologic abnormalities were detected more commonly in lame horses. Because of its heterogeneity and small thickness, evaluation of the DSIL in MRI can be challenging. The aim of the study was to determine the optimal sequence and the ideal transverse perpendicular angle for visualization of the DSIL before and after arthrography of the distal interphalangeal joint (DIPJ). Twenty-five cadaver forelimbs were examined with low-field MRI. Sagittal, frontal, and three different angled transverse planes were obtained before and after arthrography of the DIPJ. All planes were acquired in T1w (weighted) Gradient Recall Echo (GRE), T2∗w GRE, T2w Fast Spin Echo (FSE), and Short Tau Inversion Recovery (STIR) FSE and visualization of the DSIL was scored by two observers. Visualization of the DSIL was best on sagittal T2w FSE and STIR FSE images. All transverse planes were inferior compared with sagittal sequences. After arthrography of the DIPJ, visualization of the DSIL origin improved in sagittal T2w FSE sequences, and agreement between observers increased for sagittal T2w FSE and STIR FSE images. Sagittal T2w FSE and STIR FSE images allowed good visualization of the DSIL in low-field MRI. Visualization of the DSIL did not improve for altered angled transverse sequences but increased with arthrography of the DIPJ. Subjective influence between different observers was found but decreased with DIPJ arthrography.

ZusammenfassungEine 17 Monate alte Färse der Rasse Deutsch-Holstein wurde wegen seit einem Tag bestehender Schluckstörungen, geringgradiger Lähmung der Zunge sowie Lidschwellungen vorgestellt. Die klinische Untersuchung ergab eine gestreckte Kopf-Hals-Haltung, hochgradige Salivation, einen reduzierten Zungentonus sowie einen Schlotterkiefer mit vollständigem Ausfall des Schluckreflexes. Am rechten Auge zeigten sich ein herabhängend wirkendes Oberlid, mukopurulenter Ausfluss, Expositionskeratitis, Hornhauttrübung und Miosis. Linksseitig bestanden ein mittelgradiger Exophthalmus und geringgradiger muköser Nasenausfluss. Bei der neurologischen Untersuchung waren multiple Kopfnervenausfälle festzustellen, die für das Vorliegen eines Hirnbasissyndroms sprachen. Röntgenologisch ließ sich ventral des Siebbeins eine klar abgegrenzte, weichteildichte Verschattung von ca. 5 cm Durchmesser mit Verdacht auf intraneurokranialen Ursprung nachweisen, die sich rhinoskopisch mit unebener, geröteter, teils von eitrigem Exsudat bedeckter Oberfläche darstellte. Die pathomorphologische Untersuchung des euthanasierten Rindes bestätigte das Vorliegen einer Neoplasie, die von der Fossa hypophysialis des Corpus ossis basisphenoidalis bis in den Bereich ventral des Siebbeins reichte. Die histopathologischen Befunde passten zu einem sogenannten „kleinzelligen blauen Rundzelltumor“.

Zusammenfassung Ziel der Studie war, die diagnostische Aussagekraft der mittels Niederfeld- Magnetresonanztomographie (MRT) erhobenen Befunde bei septischen Erkrankungen im Hufbereich nach durchdringenden Verletzungen (Nageltritt) zu überprüfen. Material und Methoden: Bei 10 Pferden mit Verletzungen im Hufbereich wurden die MRT-Befunde mittels klinischer, chirurgischer, röntgenologischer, sonographischer und computertomographischer Befunde und/oder postmortaler histologischer Untersuchung validiert. Ergebnisse: Am genauesten ließ sich eine Beteiligung des Strahlbeins nachweisen, die am besten in der fettunterdrückten Sequenz (Short-Tau-Inversion-Recovery-Sequenz, STIR) ermittelt wurde. Defekte der tiefen Beugesehne waren ausgezeichnet nachweisbar, aber unspezifischer als Veränderungen des Strahlbeins. Sie ließen sich am besten in transversalen T2-gewichteten Fast-Spin- Echo-Sequenzen (T2w FSE) nachvollziehen. Der Stichkanal war in sämtlichen Fällen in allen Ebenen erkennbar. Auch hier erwies sich die T2w FSE als gut geeignet. Am wenigsten deutlich konnte die septische Bursitis nachvollzogen werden. Eine Erkrankung der Bursa war am besten in der sagittalen Ebene erkennbar. Schlussfolgerung und klinische Relevanz: Die MRT-Diagnostik von Erkrankungen im Hufbereich nach Verletzung durch Fremdkörper (vor allem Nageltritt) stellt eine zuverlässige Untersuchungsmethode zur Absicherung der Diagnose dar. Dabei eignet sich eine transversale T2w FSE am besten zum Nachweis eines Stichkanals und Sehnendefekts. Für die Diagnostik der Bursitis sind der Stichkanalverlauf und sekundäre Reaktionen des Strahlbeins ausschlaggebend. Fettunterdrückte Sequenzen können eine Knochenbeteiligung gut nachweisen, wenn der Stichkanal nicht direkt bis auf den Knochen zu verfolgen ist. Die beschriebenen Fälle verdeutlichen, dass bei durchdringenden Verletzungen im Hufbereich eine MRT-Untersuchung angezeigt sein kann, wenn die Beteiligung von Strukturen vermutet wird, die eine schlechte Prognose bedingen. Nur die MRT erlaubt intravital eine Beurteilung der verschiedenen Strukturen innerhalb der Hornkapsel. Diese Information wie derum ist essenziell bei der Entscheidungsfindung für eine gezielte Therapie bzw. einer Vermeidung unnötiger Behandlungen.

In human chronic lymphocytic leukemia (CLL) pathogenesis, B-cell antigen receptor signaling seems important for leukemia B-cell ontogeny, whereas the microenvironment influences B-cell activation, tumor cell lodging, and provision of antigenic stimuli. Using the murine Eμ-Tcl1 CLL model, we demonstrate that CXCR5-controlled access to follicular dendritic cells confers proliferative stimuli to leukemia B cells. Intravital imaging revealed a marginal zone B cell–like leukemia cell trafficking route. Murine and human CLL cells reciprocally stimulated resident mesenchymal stromal cells through lymphotoxin–β-receptor activation, resulting in CXCL13 secretion and stromal compartment remodeling. Inhibition of lymphotoxin/lymphotoxin–β-receptor signaling or of CXCR5 signaling retards leukemia progression. Thus, CXCR5 activity links tumor cell homing, shaping a survival niche, and access to localized proliferation stimuli. Significance: CLL and other indolent lymphoma are not curable and usually relapse after treatment, a process in which the tumor microenvironment plays a pivotal role. We dissect the consecutive steps of CXCR5-dependent tumor cell lodging and LTβR-dependent stroma–leukemia cell interaction; moreover, we provide therapeutic solutions to interfere with this reciprocal tumor–stroma cross-talk. Cancer Discov; 4(12); 1448–65. ©2014 AACR. See related commentary by López-Guerra et al., p. 1374 This article is highlighted in the In This Issue feature, p. 1355

Dental disorders are one of the top-ranking clinical domains in equine practice. Scintigraphy has emerged as a useful diagnostic imaging modality for equine dental diseases. There is a paucity of the normal scintigraphic uptake and its correlation with age. This prospective, cross-sectional, descriptive and pilot-designed study aimed to describe the radioisotope uptake (RU) patterns of the reserved crown and periodontal bone of the maxillary and mandibular cheek teeth (CT) in clinically sound horses and to evaluate the age effect on RU. For this purpose, 60 horses that underwent a bone scintigraphy for reason unrelated to head were included and divided equally into four age groups. Regions of interests (ROIs) were positioned around alveolar and periodontal bone of each maxillary and mandibular CT including their reserve crown, and a reference ROI positioned at the mandibular ramus. The count per pixel of each ROI was measured using a dedicated software and the RU ratio relative to the reference region was calculated. The results showed that the maxillary and mandibular CT had a standard RU pattern, where it increased from rostral to caudal, and peaked in the middle of dental arcades and decreased slightly towards the last CT. The maxillary CT had a higher RU compared with the mandibular CT, and there was no significant difference in the maxillary and mandibular CT uptake between age groups. This information may aid veterinarians evaluating potentially abnormal dental scintigraphy.

Sheep are commonly used as a model for human spinal orthopaedic research due to their similarity in morphological and biomechanical features. This study aimed to document the volumes of vertebral bodies and compare the generated results as well as morphometry of the sheep lumbar spine to human published data. For this purpose, computed tomography scans were carried out on five adult Merino sheep under general anaesthesia. Transverse 5 mm thick images were acquired from L1 to L6 using a multi-detector-row helical CT scanner. Volume measurements were performed with dedicated software. Four spinal indices and Pavlov’s ratio were calculated. Thereafter, the generated data were compared to published literature on humans. The mean vertebral body volume showed an increase towards the caudal vertebrae, but there were no significant differences between the vertebral levels (P>0.05). Compared to humans, sheep vertebral body volumes were 48.6% smaller. The comparison of absolute values between both species revealed that sheep had smaller, longer and narrower vertebral bodies, thinner intervertebral discs, narrower spinal canal, longer transverse processes, shorter dorsal spinous processes and narrower, higher pedicles with more lateral angulations. The comparison of the spinal indices showed a good similarity to human in terms of the vertebral endplates and spinal canal. The results of this study may be helpful for using the sheep as a model for human orthopaedic spinal research if anatomical differences are taken into account.

Six cadaver pelvic limbs were obtained from clinically sound dromedary camels and examined radiographically and ultrasonographically using a 7.5 MHz convex transducer. Radiographic examination was performed in dorsoplantar, lateromedial, dorsolateral-plantaromedial oblique and plantarolateral-dorsomedial oblique projections, and the bony structures and articulations of the tarsal joint were outlined. The tarsus was ultrasonographically investigated in four planes (dorsal, medial, lateral and plantar), and each plane was scrutinized in four levels (calcaneal tuber, tibial malleoli, base of calcaneus and proximal end of metatarsus) in both transverse and longitudinal views. Limbs were examined grossly, frozen at -20°C and sectioned. Radiographic and ultrasonographic findings correlated well with the gross anatomy and frozen sections. The normal appearance of bony and soft structures of the tarsus described in this study provided basic reference data for ultrasonographic and radiographic investigations of tarsal disorders in the dromedary camel.

Summary Reasons for performing study Reductions in distances between dorsal spinous processes on radiographs are used as criteria for the diagnosis of impingement of the thoracic dorsal spinous processes in horses but are potentially altered by spine motion and different head and neck positions. Objectives To determine the influence of head and neck positions on intervertebral distances between dorsal spinous processes on radiographs of thoracic spines of clinically sound horses. Methods Lateral–lateral radiographs were obtained from 23 horses in 3 head and neck positions. The width of the thoracic dorsal spinous processes and intervertebral distances between adjacent thoracic dorsal spinous processes were measured at points perpendicular to a tangent between the dorsal spinous processes and the caudal extremity of the thoracic vertebrae. Results A low head and neck position increased intervertebral distances between adjacent thoracic dorsal spinous processes from the 8th to 15th dorsal spinous processes whereas a high head and neck position had the opposite effect (P

The purpose of this study was to provide a detailed computed tomographic (CT) anatomic reference for the dromedary camel tarsus. Six cadaver pelvic limbs, obtained from three clinically and radiographically sound dromedary camels, were scanned in both soft tissue and bone windows starting from the calcaneal tuber towards the proximal metatarsus. Limbs were frozen at -20°C and sectioned transversely via an electric bone saw. The CT images were evaluated and correlated with their corresponding cryosections. The resulting images provided detailed anatomic features for bones, joints and soft tissue components of the tarsus and are intended to serve as a basic reference for the CT scanning of the dromedary camel tarsal pathology.

A retrospective analysis of 619 upper and lower cheek teeth from 62 horses was performed. Based on clinical findings, as well as radiographic and magnetic resonance (MR) imaging findings, the teeth were classified into five groups. There were 20 teeth with abnormal MR imaging signals as well as clinical alterations and 599 healthy teeth. Using MR imaging, the appearance of pulp in diseased and disease-free teeth was compared, and the appearance of abnormal pulp was studied. Subsequently, the ability of MR imaging to diagnose pulpitis and pulp necrosis in teeth with normal external appearance was investigated. In horses with clinically verified dental disease, abnormal MR imaging findings were confirmed in the pulp of all affected teeth. An enlarged blurred pulp image with a lower signal intensity was observed only in clinically diseased teeth and was a reliable criterion for diagnosing dental disease on MR imaging. On the other hand, partial or complete absence of pulp in all MR imaging sequences was observed in both diseased and nondiseased teeth. These data demonstrate that pulp changes in equine cheek teeth can be evaluated using MR imaging.