IL-27 maintains cytotoxic Ly6C+ γδ T cells that arise from immature precursors.

In mice, γδ-T lymphocytes that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage during thymic development. In the periphery, these cells play a critical role in host defense and anti-tumor immunity. Unlike αß-T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ-T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that CD27+ Ly6C- cells convert into CD27+Ly6C+ cells, and these CD27+Ly6C+ cells control cancer progression in mice, while the CD27+Ly6C- cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ-T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27+Ly6C+ cells and human Vδ2+ cells, while IL-27 is dispensable for mouse CD27+Ly6C- cell and human Vδ1+ cell functions. These data reveal increased complexity within IFNγ-producing γδ-T cells, comprising immature and terminally differentiated subsets, that offer new insights into unconventional T-cell biology.

Absent expansion of AXIN2+ hepatocytes and altered physiology in Axin2CreERT2 mice challenges the role of pericentral hepatocytes in homeostatic liver regeneration.

BACKGROUND & AIMS: Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail. METHODS: We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. RESULTS: We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/ß-catenin signalling and related metabolomic disturbance. CONCLUSIONS: We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci. IMPACT AND IMPLICATIONS: Understanding the source of cells which regenerate the liver is crucial to harness their potential to regrow injured livers. Herein, we show that cells which were previously thought to repopulate the liver play only a limited role in physiological regeneration. Our data helps to reconcile differing conclusions drawn from results from a number of prior studies and highlights methodological challenges which are relevant to preclinical models more generally.

Lateral manus translation for limb-sparing surgery in 18 dogs with distal radial osteosarcoma in dogs.

OBJECTIVE: To describe and report outcomes after lateral translation of the manus for limb-sparing management of distal radial osteosarcoma in dogs. STUDY DESIGN: Retrospective case series. STUDY POPULATION: Eighteen client-owned dogs. METHODS: The distal aspect of the affected radius and associated neoplastic tissues were excised. The distal aspect of the ulna was preserved except for its medial cortex, which was removed en bloc with the radial segment. The manus was translated laterally to place the radial carpal bone in contact with the distal aspect of the ulna. A limb-sparing or locking compression plate was placed on the remaining proximal radius and the 3rd metacarpal bone. A 3.5-mm SOP (string of pearls) plate was placed on the lateral aspect of the proximal ulna and the 4th metacarpal bone. Dogs were administered chemotherapy. Data were collected to assess surgical and oncologic outcomes. Limb function was subjectively assessed. RESULTS: The percentage of radius removed ranged from 43% to 94% (median 54%). Complications developed in 12 limbs, with infection in 10, biomechanical complications in 6, and local recurrence in 4. Limb function was subjectively assessed as acceptable. Median disease-free interval was 219 days, and median survival time was 370 days. CONCLUSION: Outcomes after lateral translation of the manus compared favorably to other limb-sparing techniques for dogs with distal radial osteosarcoma, particularly in dogs requiring excision of a large segment of the radius. CLINICAL SIGNIFICANCE: The lateral manus translation provides an alternative limb-sparing technique that does not require an allograft, endoprosthesis, or autograft.

Long-term outcome of dogs treated with ulnar rollover transposition for limb-sparing of distal radial osteosarcoma: 27 limbs in 26 dogs.

OBJECTIVE: To determine outcomes in dogs with distal radial osteosarcoma treated with ulnar rollover transposition (URT) limb-sparing surgery including: viability of the ulnar graft, complications, subjective limb function, disease-free interval (DFI), and survival time (ST). STUDY DESIGN: Retrospective case series. ANIMALS: Twenty-six client-owned dogs with distal radial osteosarcoma and no involvement of the ulna. METHODS: Data of dogs treated with URT were collected at the time of surgery and retrospectively from medical records and by contacting owners and referring veterinarians. RESULTS: URT technique was performed on 27 limbs in 26 dogs. The ulnar graft was determined to be viable in 17 limbs, nonviable in 3, and unknown in 7. Complications occurred in 20 limbs. Infection was diagnosed in 12 limbs. Biomechanical complications occurred in 15 and local recurrence in 2 limbs. Limb function graded by veterinarians or owners was poor in 2 limbs, fair in 4, good in 14, excellent in 3, and unknown in 4. Median DFI was 245 days and median ST was 277 days. CONCLUSION: The URT technique maintained the viability of the ulnar graft. The complication rate was high but limb function appeared acceptable. Although sufficient length of the distal aspect of the ulna must be preserved to perform this technique, local recurrence was not increased compared to other limb-sparing techniques when cases were appropriately selected.

Crystal structure of the Leishmania major MIX protein: a scaffold protein that mediates protein-protein interactions.

Infection by Leishmania and Trypanosoma causes severe disease and can be fatal. The reduced effectiveness of current treatments is largely due to drug resistance, hence the urgent need to develop new drugs, preferably against novel targets. We have recently identified a mitochondrial membrane-anchored protein, designated MIX, which occurs exclusively in these parasites and is essential for virulence. We have determined the crystal structure of Leishmania major MIX to a resolution of 2.4 Å. MIX forms an all α-helical fold comprising seven α-helices that fold into a single domain. The distribution of helices is similar to a number of scaffold proteins, namely HEAT repeats, 14-3-3, and tetratricopeptide repeat proteins, suggesting that MIX mediates protein-protein interactions. Accordingly, using copurification and mass spectroscopy we were able to identify several proteins that may interact with MIX in vivo. Being parasite specific, MIX is a promising new drug target and, thus, the structure and potential interacting partners provide a basis for structure-guided drug discovery.

Hemipelvectomy in dogs and cats: technique overview, variations, and description.

OBJECTIVE: To review and describe the surgical procedure of hemipelvectomy in dogs and cats. STUDY DESIGN: Descriptive report. ANIMALS: Dogs and cats. METHODS: Anatomic surgical technique description and PubMed literature search. RESULTS: Hemipelvectomy is an involved, aggressive surgical procedure that must be tailored to each specific clinical case. Thorough preoperative assessment and planning, as well as considerations for closure options are critical for a successful outcome. Although outcome data are scant, the prognosis after hemipelvectomy correlates to the underlying disease process. Proactive postoperative analgesia and slower return to function are important factors in patient management. CONCLUSIONS: With careful pre- and postoperative planning, hemipelvectomy can be used successfully in dogs and cats. CLINICAL RELEVANCE: Hemipelvectomy in the dog and cat is a viable treatment option for selected neoplastic and traumatic conditions.

Adverse effect of complete thoracic evacuation during chronic pneumothorax in a dog.

OBJECTIVE: To describe an unusual response to evacuation of a chronic pneumothorax in a dog. CASE: Resolution of a pneumothorax is usually associated with lung expansion and a marked improvement in pulmonary function. In the case presented here, evacuation of a chronic pneumothorax improved oxygenation, but markedly impaired ventilation. CLINICAL RELEVANCE: This is a previously unreported complication associated with the treatment of pneumothorax in dogs. It suggests that in some cases of chronic pneumothorax, total evacuation of the pleural air pocket may improve oxygenation but ventilation deteriorates and increases PCO2, causing further complications.

Tolerance of cutaneous or mucosal flaps placed into a radiation therapy field in dogs.

OBJECTIVE: To determine the clinical outcome and factors affecting cutaneous or mucosal flaps in dogs treated with radiation therapy (RT). STUDY DESIGN: Longitudinal clinical study. ANIMALS: Twenty-six client-owned dogs. METHODS: Dogs entered in the study had a flapping procedure and RT as part of their treatment. The sequence of flapping and RT included: (1) planned preoperative RT, (2) postoperative RT, and (3) flapping as a salvage procedure for management of complications or local tumor recurrence after RT. Flap complications were defined as necrosis, local infection, dehiscence, and ulceration. The risk and severity of flap complication were analyzed independently. RESULTS: Twenty (77%) dogs had a complication; 6 dogs required an additional flapping procedure; and 4 dogs had an unresolved complication. Flapping procedures performed to correct a complication, or failure of RT, had a significantly greater risk for complication; however, postoperative RT decreased the severity of complication. A dose per fraction of 4 Gy compared with 3 Gy was prognostic for increased severity of complications, whereas the head and neck location was prognostic for decreased severity of complication. CONCLUSIONS: Although morbidity was substantial, cutaneous or mucosal flaps were used successfully in an RT field in 85% of the dogs. Flaps that were part of the planned therapy as opposed to those used to correct a complication or failure of RT had a better clinical outcome. CLINICAL RELEVANCE: Cutaneous or mucosal flaps can be part of the treatment of dogs with tumor when adjuvant or neoadjuvant RT is to be used.

Evaluation of the effect of routine histologic processing on the size of skin samples obtained from dogs.

OBJECTIVE: To determine the effects that routine histologic processing has on the dimensions of samples of normal skin of dogs and assess whether the inclusion of a muscle or fascial layer in such samples alters those effects. SAMPLE POPULATION: Skin samples obtained from 6 medium-sized adult dogs with grossly normal skin. PROCEDURE: From each dog, skin samples (with or without underlying fascia or muscle) were obtained from 3 sites bilaterally (6 samples/dog) and processed routinely for histologic evaluation; their dimensions were measured at intervals during the experiment. RESULTS: As a result of processing, skin samples decreased in size (combined percentage change in length and width) and increased in thickness, compared with their original dimensions. Samples without fascia or muscle decreased in size by 21.1% to 32.0% and increased in thickness by 45.1 % to 75.8%. The site of sample origin influenced processing-associated changes in sample size but did not affect the change in thickness. Decreases in dimensions did not vary with inclusion of fascia but did vary with inclusion of muscle. The change in thickness did not vary with inclusion of a layer of fascia or muscle. CONCLUSIONS AND CLINICAL RELEVANCE: Processing of skin samples obtained from dogs for histologic evaluation can cause changes in sample dimensions; samples may decrease in length and width by as much as 32% and increase in thickness by 75.8%, compared with their original dimensions. The presence of muscle in canine skin samples can restrict the amount of shrinkage in length or width associated with processing.

Biomechanical comparison of cortical radial graft versus ulnar transposition graft limb-sparing techniques for the distal radial site in dogs.

OBJECTIVE: To compare the biomechanical properties of radial graft (RG) versus ulnar transposition graft (UTG) limb-sparing techniques in the dog. STUDY DESIGN: Cadaveric study. ANIMALS: Six pairs of normal canine thoracic limbs. METHODS: In each pair of limbs, 1 limb was subjected to the RG technique and the other to the UTG technique. Limbs were tested in axial loading until failure. Modes of failure and biomechanical properties were compared between the 2 groups. Percent coverage of the metacarpal bone by the plate was retrospectively compared between the limbs that failed by fracture of the metacarpal bones and those that did not. RESULTS: RG limbs had significantly greater stiffness, yield load, maximum load, maximum energy, and post-yield energy. All UTG limbs failed by cranial bending of the plate. Half of the RG limbs failed by caudal bending of the plate and half by fracture of the third metacarpal bone at the distal end of the plate. Limbs with <80% plate coverage of the metacarpal bone were significantly more likely to fail by metacarpal fracture. CONCLUSIONS: The RG technique was biomechanically superior to the UTG technique under the conditions studied. At least 80% plate coverage of the metacarpal bone should be achieved. CLINICAL RELEVANCE: Exercise restriction and coaptation bandages should be considered for patients undergoing the UTG technique to minimize potential biomechanical complications.

Use of an ipsilateral vascularized ulnar transposition autograft for limb-sparing surgery of the distal radius in dogs: an anatomic and clinical study.

OBJECTIVE: To develop a surgical technique for using the distal aspect of the ulna as a transposition autograft in a distal radial defect and to assess patency of vascular supply and viability of the distal ulna in a heterotopic position. STUDY DESIGN: Cadaveric study and clinical cases. ANIMALS: Twenty-two normal canine thoracic limbs; 3 dogs with distal radial osteosarcoma. METHODS: The arteries and veins of 12 limbs were injected with latex. Barium sulfate suspension was injected into the brachial artery of 10 other limbs after removal of the distal radius only (n = 2), ulna transposition (UT) (n = 6), or no procedure (n = 2). The distal ulna grafts were then harvested and decalcified in formic acid. The grafts were cut into 3-5 mm transverse sections and radiographed with a nonscreen film system to determine filling of intramedullary vessels with barium suspension. UT was performed in 3 dogs with distal radial osteosarcoma. Bone scintigraphy was performed 2-7 days after surgery to assess viability of the transposed ulna graft. RESULTS: Angiography confirmed patency of the caudal interosseous artery in all but 2 limbs in which the UT technique was performed; however, barium-filled vessels were identified in the medullary cavity of all ulnar grafts. Scintigraphy confirmed graft viability in the 3 dogs, all of which had good to excellent limb function. CONCLUSIONS: The distal aspect of the canine ulna can be used as a vascularized transposition autograft to replace distal radial defects, and viability can be maintained. CLINICAL RELEVANCE: The UT technique appears to be an acceptable limb-sparing technique for dogs with tumors of the distal aspect of the radius.

Accuracy of magnetic resonance imaging for estimating intramedullary osteosarcoma extent in pre-operative planning of canine limb-salvage procedures.

The objective of this work was to compare the accuracy of radiographs and magnetic resonance imaging (MRI) for estimating appendicular osteosarcoma margins. The accuracy of computed tomography (CT) and bone scintigraphy was also assessed when these studies were available. Eight dogs with appendicular osteosarcoma underwent radiographic and MRI of affected limbs. In addition, bone scintigraphy was performed in six dogs and CT examination was performed in five dogs. Two observers jointly measured tumor length on all imaging studies. Correlative gross and histologic evaluation of all affected limbs was performed to determine tumor extent as measured from the nearest articular surface. Results from imaging studies were compared to gross and microscopic morphometry findings to determine the accuracy of each modality for determining tumor boundaries. MRI images were accurate with a mean overestimation of actual tumor length of 3 +/- 13%. T1-weighted non-contrast images were superior in identifying intramedullary tumor margins in most instances whereas contrast-enhanced images provided supplemental information in two dogs. Lateromedial and craniocaudal radiographs overestimated tumor length by 17 +/- 28% and 4 +/- 26%, respectively. Scintigraphy and CT overestimated tumor margins by 14 +/- 28% and 27 +/- 36%, respectively. MRI appears to be an accurate diagnostic imaging modality in determining intramedullary osteosarcoma boundaries. MRI should be considered as part of a pre-operative assessment of appendicular osteosarcoma, particularly when a limb-sparing procedure is contemplated.