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An 11-year-old Doberman Pinscher with clinical and histological skin features consistent with colour dilution alopecia had a long history of skin disease. The animal had hundreds of papules affecting the ‘blue’ haired areas of the hair coat. The predominant skin lesions included keratinizing infundibular cysts, keratin horns, perifollicular dermatitis, and sebaceous hyperplasia; these lesions were often accompanied by furunculosis and suppurative panniculitis. Over a 9-month period, 32 lesions were examined histologically, which included two lipomas, one infiltrative lipoma, one squamous papilloma, two mast cell tumours, four cavernous haemangiomas, one haemangiosarcoma, and three melanomas. It is unknown at present whether the colour dilution phenotype confers increased risk for tumours of the skin and or subcutaneous tissues. Resume— Un doberman de 11 ans avec des manifestations cutanees cliniques et histologiques compatibles avec le diagnostic d'alopecie des couleurs diluees avait une longue histoire de problemes cutanes. L'animal presentait des centaines de papules affectant les zones de couleur bleue. Les lesions cutanees predominantes consistaient en kystes infundibulaires keratinisants, cornes cutanees, dermatite perifolliculaire et hyperplasie sebacee; ces lesions s'accompagnaient souvent de furonculose et de panniculite suppurative. Sur une periode de 9 mois, 32 lesions ont fait l'objet d'un examen histologique, dont deux lipomes, un lipome infiltrant, un papillome, deux mastocytomes, quatre hemangiomes caverneux, un hemangiosarcome, et trois melanomes. On ne sait actuellement pas si le phenotype couleur diluee constitue un facteur de risque accru pour les tumeurs de la peau et des tissus sous cutanes. [Madewell, B. R., Ihrke, P. J., Griffey, S. M. Multiple skin tumours in a Doberman Pinscher with colour dilution alopecia. (Tumeurs cutanees multiples chez un doberman presentant une alopecie des couleurs diluees.) Veterinary Dermatology 1997; 8: 59–62.] Resumen Un doberman pinscher de 11 anos con caracteristicas clinicas e histologicas compatibles con alopecia de color diluido tenia una larga historia de enfermedad cutanea. El animal presentaba centenares de papulas afectando las areas de pelo “azul”. Las lesiones predominantes incluian quistes infundibulares queratinizados, cuernos de queratina, dermatitis perifolicular e hiperplasia sebacea; estas lesiones se acompanaban de furunculosis y paniculitis supurativa. Durante un periodo de 9 meses, 32 lesiones fueron examinadas histologicamente, incluyendo 2 lipomas, un lipoma infiltrativo, un papiloma escamoso, dos mastocitomas, cuatro hemangiomas cavernosos, un hemangiosarcoma y tres melanomas. Se desconoce aun si el fenotipo de color diluido aumenta el riesgo para la aparicion de tumores en tejidos cutaneos y subcutaneos. [Madewell, B. R., Ihrke, P. J., Griffey, S. M. Multiple skin tumours in a Doberman Pinscher with colour dilution alopecia. (Multiples tumores cutaneos en un Doberman Pinscher con alopecia de color diluido.) Veterinary Dermatology 1997; 8: 59–62.] Zusammenfassung— Ein elfjahriger Dobermann mit klinischen und histologischen Befunden, die bei Farbmutantenalopezie auftreten, wies eine lange Vorgeschichte mit Hautkrankheiten auf. Das Tier zeigte Hunderte von Papeln, die die “blau” behaarten Bezirke des Felles betrafen. Die vorherrschenden Hautveranderungen bestanden in keratinisierenden infundibularen Zysten, Keratinhornern, perifollikularer Dermatitis und Talgdrusenhyperplasie; diese Veranderungen wurden oft von Furunkulose und eitriger Pannikulitis begleitet. Uber eine Zeitspanne von 9 Monaten wurden 32 Veranderungen histologisch untersucht, wovon zwei in Lipomen bestanden, eine in einem infiltrativen Lipom, eine in einem squamosen Papillom, zwei in Mastzelltumoren, vier in kavernosen Hamangiomen, eine in einem Hamangiosarkom und drei in Melanomen. Es ist zur Zeit nicht bekannt, ob der Phanotyp mit der Farbverdunnung mit einem erhohten Risiko fur Tumoren der Haut und/oder des subkutanen Gewebes einhergeht. [Madewell, B. R., Ihrke, P. J., Griffey, S. M. Multiple skin tumours in a Doberman Pinscher with colour dilution alopecia. (Multiple Hauttumoren bei einem Dobermann mit Farbmutantenalopezie.) Veterinary Dermatology 1997; 8: 59–62.]

Summary: Gross necropsy with documentation of changes present. Collection and histologic processing (paraffin blocks/H&E slides) and interpretation of kidneys, liver, spleen, pancreas, heart, lungs, esophagus, trachea, thymus, mesenteric lymph nodes, GI Tract, cerebrum, cerebellum, urinary bladder, and reproductive tract and additional tissues with gross changes. This includes photodocumentation of significant findings. This assay can be customized to include other target organs based on findings from other MMPC assays. This procedure is to be followed by trained Comparative Pathology Laboratory (CPL) staff responsible for performing necrospy diagnostics procedures at CPL.

Acute pancreatitis (AP) is a common and devastating gastrointestinal disorder that causes significant morbidity. The disease starts as local inflammation in the pancreas that may progress to systemic inflammation and complications. Protein tyrosine phosphatase 1B (PTP1B) is implicated in inflammatory signaling, but its significance in AP remains unclear. To investigate whether PTP1B may have a role in AP, we used pancreas PTP1B knockout (panc-PTP1B KO) mice and determined the effects of pancreatic PTP1B deficiency on cerulein- and arginine-induced acute pancreatitis. We report that PTP1B protein expression was increased in the early phase of AP in mice and rats. In addition, histological analyses of pancreas samples revealed enhanced features of AP in cerulein-treated panc-PTP1B KO mice compared with controls. Moreover, cerulein- and arginine-induced serum amylase and lipase were significantly higher in panc-PTP1B KO mice compared with controls. Similarly, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B, IL-6, and tumor necrosis factor-α were increased in panc-PTP1B KO mice compared with controls. Furthermore, panc-PTP1B KO mice exhibited enhanced cerulein- and arginine-induced NF-κB inflammatory response accompanied with increased mitogen-activated protein kinases activation and elevated endoplasmic reticulum stress. Notably, these effects were recapitulated in acinar cells treated with a pharmacological inhibitor of PTP1B. These findings reveal a novel role for pancreatic PTP1B in cerulein- and arginine-induced acute pancreatitis.

Beginning in 2015, athymic nude sentinel mice from conventional, medium-, and high-security facilities presented to the Comparative Pathology Laboratory (CPL) with weight loss, diarrhea, and/or rectal prolapse. Regardless of whether clinical signs were present or absent, the gross observation of ceco-colonic thickening corresponded histologically to pleocellular typhlocolitis with mucosal hyperplasia and lamina proprial multinucleated cells. A subset of affected sentinels exhibited granulomatous serositis and hepatosplenic necrosis with multinucleated cells. Initial suspicion of mouse hepatitis virus infection was excluded by polymerase chain reaction, electron microscopy, and serology. Multinucleated giant cells were confirmed as macrophages by positive immunoreactivity to Mac-3 and Iba-1 and negative immunoreactivity to pancytokeratin. From conventional and medium-security facilities, Helicobacter species were identified in 40 of 143 (27.9%) mice, with H. hepaticus accounting for 72.5% of identified Helicobacter species. Other agents included opportunistic bacterial infection (41/145, 28.3%), murine norovirus (16/106, 15.1%), and pinworms (2/146, 1.4%). From high-security facilities, only Enterobacter cloacae was identified (2/13, 15.4%), and no evidence of Helicobacter sp., murine norovirus, or pinworms was present. No potentially infectious disease agent(s) was identified in 71 of 146 (48.6%) affected nude sentinels from conventional and medium-security facilities and 11 of 13 (84.6%) affected nude sentinels from high-security facilities. No statistically significant differences in histologic lesion scores were identified between Helicobacter-positive and Helicobacter-negative mice. Thus, proliferative typhlocolitis with multinucleated giant cells was considered a nonspecific histologic pattern associated with a variety of primary and opportunistic pathogens in athymic nude mice.

Author(s): Moore, Bret A; Roux, Michel J; Sebbag, Lionel; Cooper, Ann; Edwards, Sydney G; Leonard, Brian C; Imai, Denise M; Griffey, Stephen; Bower, Lynette; Clary, Dave; Lloyd, KC Kent; Herault, Yann; Thomasy, Sara M; Murphy, Christopher J; Moshiri, Ala | Abstract: PurposeThe purpose of this study was to quantify the frequency and severity of ocular abnormalities affecting wild-type C57BL/6N mice, the most common strain used worldwide for the creation of single-gene knockouts.MethodsA total of 2773 animals (5546 eyes) were examined at one colony at UC Davis and in three more colonies at the Institut Clinique de la Souris in Strasbourg, France. Mice were examined at 15 to 16 weeks postnatal age by performing anterior segment biomicroscopy, posterior segment examination by indirect ophthalmoscopy, intraocular pressure measurement, and optical coherence tomography of anterior and posterior segment structures.ResultsCommon ocular findings in the C57BL/6N strain included corneal deposits (3%), increased optical density of the anterior lens capsule (67%), punctate nuclear cataracts (98%), vitreous crystalline deposits (61%), hyaloid vascular remnant (6%), and retinal dysplasia attributed to the rd8 mutation (58%). Interestingly, retinal dysplasia was more common in male mice in all four breeding colonies evaluated in this study. The thickness of ocular tissues and compartments were measured by spectral-domain optical coherence tomography, including the central cornea, anterior chamber, vitreous, and retinal layers. Intraocular pressure was measured by rebound tonometry.ConclusionsOcular abnormalities are common in anterior and posterior segments of the C57BL/6N mouse, the most common background on which single-gene knockout mice have been made. It is important that vision scientists understand the extent and variability of ocular findings associated with this particular genetic background of mice.

The voltage-gated potassium channel Kv1.3 has been recently identified as a molecular target that allows for selective pharmacological suppression of effector memory T (T(EM)) cells without affecting the function of naïve and central memory T cells. We here investigated whether PAP-1, a small molecule Kv1.3 blocker (EC50=2 nM), could suppress allergic contact dermatitis (ACD). In a rat model of ACD, we first confirmed that the infiltrating cells in the elicitation phase are indeed CD8+ CD45RC- memory T cells with high Kv1.3 expression. In accordance with its selective effect on T(EM) cells, PAP-1 did not impair sensitization, but potently suppressed oxazolone-induced inflammation by inhibiting the infiltration of CD8+ T cells and reducing the production of the inflammatory cytokines IFN-gamma, IL-2, and IL-17 when administered intraperitoneally or orally during the elicitation phase. PAP-1 was equally effective when applied topically, demonstrating that it effectively penetrates skin. We further show that PAP-1 is not a sensitizer or an irritant and exhibits no toxicity in a 28-day toxicity study. Based on these results we propose that PAP-1 could potentially be developed into a drug for the topical treatment of inflammatory skin diseases such as psoriasis.

Acute pancreatitis (AP) is an inflammatory disease, and is one of the most common gastrointestinal disorders worldwide. Soluble epoxide hydrolase (sEH; encoded by Ephx2) deficiency and pharmacological inhibition have beneficial effects in inflammatory diseases. Ephx2 whole-body deficiency mitigates experimental AP in mice, but the suitability of sEH pharmacological inhibition for treating AP remains to be determined. We investigated the effects of sEH pharmacological inhibition on cerulein- and arginine-induced AP using the selective sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which was administered before and after induction of pancreatitis. Serum amylase and lipase levels were lower in TPPU-treated mice compared with controls. In addition, circulating levels and pancreatic mRNA of the inflammatory cytokines tumor necrosis factor-α, interleukin Il-1β, and Il-6 were reduced in TPPU-treated mice. Moreover, sEH pharmacological inhibition before and after induction of pancreatitis was associated with decreased cerulein- and arginine-induced nuclear factor-κB inflammatory response, endoplasmic reticulum stress, and cell death. sEH pharmacological inhibition before and after induction of pancreatitis mitigated cerulein- and arginine-induced AP. This work suggests that sEH pharmacological inhibition may be of therapeutic value in acute pancreatitis.

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+CCR7−CD45RA−effector memory T cells (TEMcells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naïve or central-memory (TCM) cells. In TEMcells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvβ2, SAP97, ZIP, p56lck, and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation, they suppress Ca2+-signaling, cytokine production, and proliferation of autoantigen-specific TEMcells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.

The Hfe( - / - ) mouse is a model for human hereditary hemochromatosis (HHH). The accumulation of tissue iron in this condition has led to the suggestion that HHH patients may be at higher risk for neurodegenerative diseases. In this study, adult male Hfe( - / - ) mice and wildtype controls ( n = 12/group) were evaluated for impairment with motor tests (stride length, landing footsplay, rotarod) as well as a general observational battery (Functional Observational Battery, FOB). Hfe( - / - ) mice were characterized by more falls from the rotarod, wider forelimb landing footsplay and hypersensitivity to proximal stimulation. Iron accumulation in brain was not detected by histopathology. These data suggest that a motor syndrome may be associated with HHH that could be further understood through the Hfe( - / - ) mouse model.

We evaluated implanted rat mammary adenocarcinoma tumors during a 5-week period using ultrasound, computed tomography (CT), and histology.Contrast-enhanced ultrasound with a destruction-replenishment imaging scheme was used to derive estimates of blood volume and flow. These ultrasound-derived measures of microvascular physiology were compared with contrast-enhanced CT-derived measures of perfusion and vascular volume made by the Mullani-Gould formula and Patlak analysis, respectively.The tumor cross-sectional area and necrotic core cross-sectional area determined by the 3 methods were correlated (r0.8, P0.001, n=15). The spatial integral of perfusion estimated by CT correlated with the spatial integral of flow from ultrasound (P0.05). The contrast-enhanced tumor area calculated from the ultrasound analysis was highly correlated with the contrast-enhanced area estimated by CT images (r=0.89, P0.001, n=15). However, the fraction of the tumor area enhanced by the CT contrast agent was significantly larger than either the fraction enhanced by ultrasound contrast agent or than the viable area as estimated from histology slides.Destruction-replenishment ultrasound provides valuable information about the spatial distribution of blood flow and vascular volume in tumors and ultrasound analysis compares favorably with a validated contrast-enhanced CT method.

Objective— To compare 3 techniques for ureteroneocystostomy in cats. Study Design— Experimental surgical study. Animals— Fifteen adult cats. Methods— Cats (15) had ureteroneocystostomy with ureteronephrectomy of the contralateral kidney: 5 cats had an intravesical mucosal apposition technique (modified Leadbetter-Politano; intravesical-MA group), 5 cats had extravesical ureteroneocystostomy (modified Lich Gregoir) using a simple continuous suture pattern (extravesical-SC group) and 5 cats had an extravesical technique using a simple interrupted suture pattern (extravesical-SI group). Renal function was evaluated by measuring serum creatinine concentration. Ultrasonographic assessment of the kidney and ureteroneocystostomy site was performed the day after surgery, twice weekly for 3 weeks and once weekly for the remainder of the study. Cats were euthanatized 50 days after surgery. The kidney and ureter removed at surgery, the remaining kidney, ureter, ureteroneocystostomy site, and bladder were examined histologically. Results— Two extravesical-SC cats were euthanatized because of azotemia and uroabdomen, and 1 died acutely at day 4 for unknown reasons. In the intravesical-MA and extravesical-SI cats, the serum creatinine concentration increased after surgery, peaking at a mean (±SD) of 9.4±2.4 mg/dL and 4.9±3.3 mg/dL on day 3, and decreasing to 3.4±5.7 mg/dL and 1.5±0.4 mg/dL on day 7, respectively. The extravesical-SI technique was associated with consistently lower serum creatinine concentrations for the first week after surgery compared with the other techniques. The mean serum creatinine concentration was within the reference range in cats in the intravesical-MA and extravesical-SI groups by days 10 and 5, respectively. Renal pelvic dilatation occurred in all cats but resolved more rapidly in cats after extravesical techniques. There was no significant difference in serum creatinine concentrations or renal pelvic dilation between the intravesical-MA and extravesical-SI techniques. Bladder mass height at the anastomosis site was significantly larger and persisted for longer with intravesical-MA technique. Conclusion— An extravesical-SI technique is seemingly the choice for ureteroneocystostomy in cats with undilated ureters. Renal pelvic dilation on ultrasound examination should be expected after ureteroneocystostomy in cats. Clinical Relevance— An extravesical ureteroneocystostomy technique using a simple interrupted pattern for anastomosis should be considered in cats undergoing renal transplantation.

Objectives— To evaluate the mechanism of vascular occlusion after placement of an ameroid constrictor (AC) on a large intra-abdominal vein and document changes in blood flow. Study Design— Experimental study. Animals— Six adult dogs. Methods— Six 6.5 mm ACs were digitally scanned to measure area and circumference of the ameroid and inner lumen before surgery. ACs were surgically positioned around the left common iliac vein (CIV) in each dog. Peri-vascular ultrasonic flow probes were positioned on the left CIV cranial to the AC, and on the right CIV as an internal control. Blood flow measurements were recorded daily until there was no, or prolonged minimal, blood flow, at which time the dogs were euthanatized. Left and right CIVs were removed for histologic evaluation, and the ACs re-scanned to evaluate degree and direction of expansion of the ameroid. Results— Reduction in blood flow occurred within 10 days in all dogs. Three dogs had complete occlusion by day 10 from thrombus formation. One dog damaged the flow probes at 13 days when blood flow was approximately 50% of intra-operative values; the dog was euthanatized at 37 days; there was partial occlusion from thrombosis. Two dogs had persistent low blood flow and occlusion from thrombus formation when euthanatized at 59 and 98 days. There was a significant increase in total AC area and outer circumference, and decrease in luminal area and inner circumference when presurgical and postocclusion measurements were compared. Conclusions— Vascular occlusion after placement of an AC on a large intra-abdominal vein occurred from thrombus formation. Clinical Relevance— A similar pattern of venous occlusion may occur after placement of an AC on a portosystemic shunt, which may lead to both short- and long-term complications associated with portal hypertension.

Pancreatolithiasis has been documented to occur naturally in humans and cattle. It has been associated with chronic pancreatitis in humans, and, when found, it may signify the presence of chronic pancreatic disease. This is the first report of a case involving a cat that had both an apparent obstruction with pancreatolithiasis as well as concurrent evidence of chronic pancreatic changes on histopathological evaluation. Additionally, this case documents the presence of a suspected congenital abnormality of a feline exocrine pancreas.

To determine the ability of contrast material-enhanced ultrasonography (US) to assess replenishment time in a rat kidney and adenocarcinoma tumor model.Mammary adenocarcinoma cells were implanted into the subcutaneous tissues of the flank of 11 rats. Resultant tumors were imaged serially with contrast-enhanced US and compared with images of the rat kidney, a highly perfused normal organ. The US acquisition and processing methods yield images of perfused tumor regions and the times required to achieve 80% replenishment. Findings at contrast-enhanced computed tomography (CT) and light microscopy of hematoxylin-eosin-stained tumor tissue were compared. Paired Student t test was performed to compare the accuracy of US with that of histologic examination and CT in the detection of viable tumor regions.Replenishment of the kidney cortex microvasculature requires 1-5 seconds compared with a replenishment time of 6-14 seconds in tumors. Over the time course of tumor growth, the mean perfusion time becomes progressively longer, and a wider range of perfusion times is detected. Comparison of findings at US, CT, and histologic examination suggested that all three methods yield correlated estimates of the percentage of viable perfused tumor cells. Results of the t test suggested that the viable tumor percentages observed at US are not significantly different from those observed at CT and histologic examination (US vs CT, P =.92; US vs histologic examination, P =.94).Repeated measurements of microvascular flow rate can be accomplished in a rat animal model with a minimally invasive technique.

Objectives— To determine whether the microemulsified formulation of cyclosporine (MCsA; Neoral; Novartis A.G.), combined with azathioprine (Imuran; Glaxo Wellcome), and prednisolone (Delta-Cortef; Upjohn), would be effective in preventing acute renal allograft rejection in unrelated mongrel dogs. To document any toxic effects associated with this drug combination. Study Design— Prospective, pilot study. Animals— Four healthy, adult, mongrel, canine renal allograft recipients. Methods— Heterotopic renal transplantation, with bilateral nephrectomy, was performed in 4 dogs. Allografts were harvested from 2 unrelated dogs that were to be euthanatized for reasons unrelated to this study. The dogs were treated for 100 days or until signs of illness or allograft rejection required euthanasia. Microemulsified cyclosporine (20 mg/kg/day), azathioprine (5 mg/kg every other day), and prednisolone (1 mg/kg/day) were administered for the prevention of acute rejection. Body weight, serum biochemistry profiles, complete blood counts, and trough whole-blood cyclosporine concentrations were measured throughout the study. Cyclosporine dose was adjusted to maintain a trough concentration of 400–500 ng/mL. Azathioprine dose was decreased if evidence of hepatotoxicity developed or if the total blood white cell count was

Intestinal intussusception is a frequent problem after experimental transplantation in dogs. This report describes the safety and efficacy of performing a modified Noble plication for the prevention of intussusception. Heterotopic renal transplantation and plication was performed in 20 dogs. Dogs were killed when the serum creatinine concentration exceeded 7 mg/dL because of acute rejection (19 dogs) or venous occlusion (1 dog). Gastrointestinal signs were commonly observed, but no dog experienced an intestinal intussusception, compared to 3 of 14 dogs (21%) previously treated using a similar immunosuppressive regimen. This study supports the routine use of enteroplication in dogs undergoing renal transplantation.

SummaryThe objective of this study was to determine whether the non-steroidal anti-inflammatory drug (NSAID) carprofen causes adverse histological changes to cartilage after prolonged administration for treatment of naturally acquired osteoarthritis.Dogs diagnosed with cranial cruciate ligament rupture and secondary osteoarthritis of their stifle joints were divided into two groups: those who had received a standard dose of carprofen for a minimum of four weeks and those who had not received a NSAID for a minimum of one month. Cartilage from the intercondylar notch was obtained when the patients underwent surgical procedures to stabilize the stifle joint. The samples were processed and evaluated for histological differences using haematoxylin and eosin and safranin-O-fast green-iron haematoxylin stains and were graded according to a Mankin scale (graded 0-14) for osteoarthritis.The histopathological findings were variable and included loss of matrix staining, irregularities to the cartilage surface, matrix fragmentation, chondrocyte clustering, matrix degeneration and vascular and synovial invasion (pannus) and were not specific to either group. There was not any significant difference between study groups (p = 0.2721) according to the Wilcoxan rank sum test.

Immunohistochemistry was used to examine feline lymphoid tumors for bcl-2 and MIB- I expression. Tumor tissues from 29 cats were selected to represent 2 groups-cats that did not respond to chemotherapy and cats that responded to therapy. Median bcl-2 immunoreactivity was 60%, and median MIB-1 reactivity was 47%. There was no significant difference in median survival time between cats with tumors with high levels of bcl-2 expression and those with low levels of expression. There was no significant difference in median survival time between cats with tumors with high levels of MIB-1 expression and those with low levels of expression. Mean bcl-2 immunoreactivity was significantly (P = .0004) higher in low-grade (73.2%) than in high-grade (16.9%) lymphomas, whereas the mean MIB-1 immunoreactivity was significantly (P = .0201) higher in high-grade (61.2%) lymphomas than in low-grade (35.0%) lymphomas. The mean bcl-2 immunoreactivity was significantly (P = .0042) greater in T-cell lymphomas (66.8%) than in B-cell lymphomas (22.8%), whereas the mean MIB-1 immunoreactivity was significantly (P = .0052) lower in T-cell lymphomas (36.4%) than in B-cell lymphomas (65.2%). Although expression of bcl-2 and MIB-1 did not appear to be linked to responses to chemotherapy in cats with lymphoma, the data suggest a possible role for these regulatory proteins in the biology of feline lymphomas.

Concurrent and sequential cisplatin-based chemoradiotherapy regimens are standard therapeutic approaches in cancer treatment. Recent clinical data suggest that these different dosing schedules may adversely affect antigen-specific immunotherapy. The goal of the present preclinical study was to explore the effects of concurrent and sequential cisplatin/radiotherapy on immune status in a lung cancer mouse model. A total of 150 C57BL/6 mice were randomized into six treatment groups: control; 8 Gy thoracic radiotherapy (dose schedules 1 and 2); cisplatin 2.5 mg/kg i.p.; cisplatin + radiotherapy (concurrent); and cisplatin + radiotherapy (sequential; n = 25, all groups). At the end of the study (week 41), serum cytokines were assessed by multiplex immunoassay, surface markers of spleen-derived lymphocytes were assessed by immunostaining and flow cytometry, lung tumor expression of programmed death ligands 1 and 2 (PD-L1/2) was evaluated by immunohistochemistry, and miRNA profiling was performed in serum and lymphocytes by quantitative real-time PCR. Lung whole mounts were prepared to assess treatment effects on lung tumor foci formation. The results showed that sequential chemoradiotherapy (two cycles of cisplatin followed by 8 Gy radiotherapy) had equivalent antitumor activity as concurrent therapy. However, sequential cisplatin/radiotherapy resulted in significant differences in several immune response biomarkers, including regulatory T cells, miR-29c, expression of costimulatory molecule CD28, and serum IFNγ. PD-L1 and PD-L2 were strongly expressed in tumor foci, but no trend was seen between groups. These results suggest that monitoring immune status may be necessary when designing treatment regimens combining immunotherapy with chemoradiotherapy. Cancer Immunol Res; 3(7); 741–50. ©2015 AACR.

Background Acute pancreatitis (AP) is a frequent gastrointestinal disorder that causes significant morbidity, and its incidence has been progressively increasing. AP starts as a local inflammation in the pancreas that often leads to systemic inflammatory response and complications. Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition in murine models has beneficial effects in inflammatory diseases, but its significance in AP remains unexplored. Methodology/Principal Findings To investigate whether sEH may have a causal role in AP we utilized Ephx2 knockout (KO) mice to determine the effects of sEH deficiency on cerulein- and arginine-induced AP. sEH expression increased at the protein and messenger RNA levels, as well as enzymatic activity in the early phase of cerulein- and arginine-induced AP in mice. In addition, amylase and lipase levels were lower in cerulein-treated Ephx2 KO mice compared with controls. Moreover, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B and IL-6 were lower in cerulein-treated Ephx2 KO mice compared with controls. Further, Ephx2 KO mice exhibited decreased cerulein- and arginine-induced NF-κB inflammatory response, MAPKs activation and decreased cell death. Conclusions -These findings demonstrate a novel role for sEH in the progression of cerulein- and arginine-induced AP.

et al., Calorie restriction (CR) without malnutrition extends life span in several animal models. It has been proposed that a decrease in the amount of polyunsaturated fatty acids (PUFAs), and especially n-3 fatty acids, in membrane phospholipids may contribute to life span extension with CR. Phospholipid PUFAs are sensitive to dietary fatty acid composition, and thus, the purpose of this study was to determine the influence of dietary lipids on life span in CR mice. C57BL/6J mice were assigned to four groups (a 5% CR control group and three 40% CR groups) and fed diets with soybean oil (high in n-6 PUFAs), fish oil (high in n-3 PUFAs), or lard (high in saturated and monounsaturated fatty acids) as the primary lipid source. Life span was increased (p, This work was supported by the National Institutes of Health (www.nia.nih.gov) (RO1AG028125).

BACKGROUND RAD is a potent immunosuppressive agent that has been shown to be effective in preventing acute and chronic allograft rejection in animal models. The HMGCoA reductase inhibitors have been found to reduce the incidence of graft vascular disease (GVD) in heart transplant patients and in animal models. This study was designed to investigate the effects of fluvastatin or pravastatin in a rodent model of GVD produced using low doses of RAD to prevent acute rejection. METHODS Hearts from Fisher 344 rats were heterotopically transplanted to Lewis rat recipients. RAD was administered orally at 0.5 mg/kg per day for days 0 to 14 and then 0.25 mg/kg per day for an additional 85 days to prevent acute rejection but allow for the development of GVD. Pravastatin (20 mg/kg per day) or fluvastatin (2 or 6 mg/kg per day) was added to the RAD treatment. At the end of a 100-day treatment period, the hearts were harvested for morphometric and histopathologic examinations. RESULTS Rats treated with fluvastatin, at either dose, had a significant (P< or =0.0239) decrease in coronary arterial intimal thickening (GVD) of approximately 43%. Rats treated with pravastatin had a 22% reduction in GVD that did not reach statistical significance. Treatment with fluvastatin, but not pravastatin, decreased the degree of endomyocardial mononuclear cell infiltration seen with RAD administered alone. CONCLUSIONS Fluvastatin significantly decreased GVD in a rat model produced using low-dose RAD immunosuppression. To a lesser extent, pravastatin also decreased GVD in this model. These data lend further support for the study of fluvastatin, pravastatin, and other HMG-CoA reductase inhibitors for the prevention of GVD in cardiac transplant patients.

The objective of this investigation was to study the effects of the first marketed haemoglobin-based oxygen carrier, Hemoglobin glutamer-200 (bovine) (Hb-200) (Oxyglobin) on splanchnic perfusion and oxygenation in a canine model of acute hypovolaemia. Twelve anaesthetized dogs [mean weight 30.8 (S.D. 1.4) kg] were instrumented for recordings of heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), cardiac output and cranial mesenteric arterial (CMA) and venous blood flows (CMV). Total and plasma haemoglobin (Hb), oxygen content and saturation, lactate concentration, pH and blood gases were analysed in arterial, mixed venous and mesenteric venous blood samples. Measurements were made before (baseline) and after 1 h of haemorrhage, after which animals were resuscitated with either shed blood (controls) or Hb-200 until HR, MAP and CVP returned to prehaemorrhage levels. Recordings were repeated immediately and 3 h after termination of fluid resuscitation, after which organ specimens were obtained for microscopic examination. Haemorrhage (average 32 ml kg(-1)) reduced MAP to 50 mm Hg, increased HR and systemic vascular resistance (SVR), and was accompanied in both the systemic and the splanchnic circulation by significant decreases in blood flow, Hb content and oxygen delivery (DO2), and lactic acidosis. In controls, all variables recovered to baseline after isovolaemic resuscitation with shed blood. In dogs resuscitated with a small volume of Hb-200 (10 ml kg(-1)), HR, MAP, CVP and CMA and CMV blood flows returned to baseline. However, cardiac output, total Hb, oxygen content and systemic and mesenteric DO2 remained depressed while SVR increased further. Mesenteric and systemic acid-base status recovered in both groups, and there was no difference in microscopic tissue damage between groups. Thus, Hb-200 reconstituted splanchnic perfusion and oxidative metabolism in spite of pronounced systemic vasoconstriction and insufficient restoration of CO and DO2; it may improve diffusive oxygen transport in the microvasculature by virtue of haemodilution and its high efficiency in the uptake and release of oxygen.

Objective—To determine whether histopathologic changes are detectable in grossly normal medial menisci from dogs with rupture of the cranial cruciate ligament (CCL).Design—Case series.Sample Population—40 medial menisci from dogs with rupture of the CCL and 20 medial menisci from control dogs without stifle joint disease.Procedure—Data evaluated included age, duration of clinical signs, and whether rupture of the CCL was complete or incomplete. Three groups (n = 20/group) were also compared on the basis of 5 histologic criteria; group-1 menisci appeared grossly normal and were obtained from dogs with naturally occurring rupture of the CCL, group-2 menisci were grossly abnormal and were also obtained from dogs with naturally occurring CCL ruptures, and group-3 menisci were collected at postmortem from dogs without stifle joint disease that were of similar age and weight as dogs in groups 1 and 2.Results—Group-2 menisci were significantly different from group-1 and -3 menisci in all histologic criteria. Group-1 menisci were significantly different from control menisci in only 1 of the 5 histologic criteria (cartilage differentiation). Dogs that were ≥ 3 years old had significantly more surface cellularity than did dogs that were < 3 years old. A significant difference was not detected between groups 1 and 2 with regard to completeness of rupture.Conclusions and Clinical Relevance—Histologic changes in meniscal cartilage correlate with gross appearance of the cartilage at time of surgery for rupture of the CCL. On the basis of minimal histologic changes, routine removal of grossly normal menisci does not appear to be warranted. (J Am Vet Med Assoc2001;218:1281–1284)

To establish the size, configuration, and histopathologic features of acute, subacute, and chronic radio-frequency (RF) electrocautery of mammary tissue in swine.Eighteen RF treatments were performed in the mammary tissue of three domestic swine under ultrasonographic (US) guidance. Histopathologic examination was performed immediately after (acute animal); 2 weeks after (subacute animal); and 4 weeks after (chronic animal) treatment.In the acute animal, lesions were firm nodules on palpation and had a distinct line of demarcation between necrotic and viable mammary tissue (mean lesion volume, 14.24 cm(3); largest volume, 29.06 cm(3)). In the subacute animal, there was diffuse coagulation necrosis with neutrophilic infiltrates at the periphery (mean lesion volume, 6.46 cm(3); largest volume, 9.47 cm(3)), and two treatment areas had a secondary bacterial infection. In the chronic animal, lesions were still palpable and firm (mean lesion volume, 11.67 cm(3); largest volume, 25.5 cm(3)), and five of six treatment sites had an area of gray to white fibrotic tissue that blended with the surrounding tissue. However, one site had a pale yellow area of central necrosis surrounded by a fibrotic area. In both the subacute and chronic animals, two and one treatment site, respectively, had minimal areas of skin necrosis.RF ablation of breast tissue is feasible in this animal model. Problems included minimal skin erythema, residual firm treatment regions at 4 weeks, slightly variable margins of coagulation necrosis, and occasional bacterial infection.

Objective—To determine quality and duration of progression-free survival (PFS) time in dogs with unresectable thyroid carcinomas treated with definitive megavoltage irradiation and analyze prognostic factors of PFS and patterns of failure (local recurrence vs metastasis). Design—Prospective clinical trial. Animals—25 dogs with locally advanced thyroid carcinomas and no evidence of metastasis. Procedure—Dogs were treated with 48 Gy during 4 weeks on an alternate-day schedule of 4 Gy/fraction. Results—Irradiation was safe and effective for treatment of large unresectable thyroid carcinomas. Progression-free survival rates were 80% at 1 year and 72% at 3 years. Time to maximum tumor size reduction ranged from 8 to 22 months. Factors affecting PFS were not found. Twenty-eight percent (7/25) of dogs developed metastasis. Dogs with bilateral tumors had 16 times the risk of developing metastases, compared with dogs with a single tumor. Dogs with no evidence of tumor progression had 15 times less risk of developing metastases. Radiation-induced hypothyroidism was suspected in 2 dogs 13 and 29 months after irradiation. Conclusions and Clinical Relevance—Irradiation is effective for local control of thyroid tumors, despite their slow regression rate. Results provided evidence that local tumor control affects metastatic outcome in dogs with thyroid carcinomas and is a strong basis for the development of new approaches that include irradiation in the management of dogs with advanced thyroid carcinomas. Improvements in local tumor control alone may be insufficient to improve survival times because of the high risk of metastatic spread before an initial diagnosis is made, which warrants initiation of early systemic treatment. (J Am Vet Med Assoc 2000;216: 1775–1779)

RATIONALE AND OBJECTIVES To characterize the long-term effects of therapeutic doses of ionizing radiation on the uptake and distribution of percutaneously delivered particulate contrast media in normal lymph nodes. METHODS Two milliliters of an iodinated nanoparticle suspension (76 mg I/mL) was injected subcutaneously or submucosally into nine normal adult beagles. Region of interest analysis was used to estimate the volume, attenuation, and iodine concentration of opacified targeted lymph nodes and nonopacifled contralateral nodes on 24-hour postinjection CT images. All lymph nodes were then irradiated with 50 Gy in 25 fractions of 2 Gy/d. Contrast-enhanced quantitative CT was repeated 12 months after irradiation. RESULTS Contrast-enhanced nodes averaged 2.3+/-0.8 times the volume of nonenhanced contralateral nodes before irradiation. The mean attenuation of contrast-enhanced nodes increased to 305 to 380 Hounsfield units from a pre-enhancement value of approximately 25 Hounsfield units. Opacified node volumes after irradiation averaged 61% to 86% of preirradiation volumes but were generally not statistically different. Contrast uptake assessed by average attenuation and iodine concentration decreased significantly by an average of 17% to 22% after irradiation and was significantly less than preirradiation uptake. Qualitatively, irradiated nodes generally appeared smaller than nonirradiated nodes, but the distribution pattern of contrast media did not appear to be appreciably altered. CONCLUSIONS Lymph node irradiation resulted in only minimal decreases in contrast media uptake and node volume at 12 months. These effects presumably would not appreciably alter the potential clinical value of indirect lymphography for evaluating patients undergoing radiation therapy.

Proliferative and apoptotic fractions of tumors were evaluated in 41 dogs with lymphoma for prediction of response to chemotherapy. All dogs had advanced clinical stage tumors, were untreated prior to study, and received identical induction-remission chemotherapy. Tumor cell proliferation was determined in all pretreatment biopsy specimens and in 18 specimens collected at the time of clinical relapse from remission. Quantitative measures included mitotic index and immunoreactivities for proliferating cell nuclear antigen (PCNA) and Ki-67. Apoptotic index was evaluated from 40 dogs pretreatment and from 16 dogs at the time of first relapse. Pretreatment tumor values for Ki-67, PCNA, and apoptosis were compared with posttreatment values. The median first relapse-free interval (RFI) and overall survival (OS) time were 174 days and 445 days, respectively. Of the proliferation markers, only the results of the Ki-67 analysis were predictive for duration of the first RFI but not OS. Pretreatment apoptotic index was also predictive of the duration of first RFI but not OS. No significant predictive value for comparison of the pretreatment and postrelapse values was demonstrated. Ki-67 labeling index and apoptotic indexes were combined to form both a proliferation/apoptotic ratio (PAR) and a sum, or turnover index. Only the PAR was predictive for duration of first RFI on multivariate analysis. Other variables that were evaluated for their influence on treatment outcome included patient age, weight, gender, clinical stage, clinical substage, and tumor immunophenotype. Of these variables, only immunophenotype was found to be of value for predicting duration of first RFI and OS.

Whole rear limbs were transplanted from Brown Norway or Lewis rat donors to Lewis rat recipients (n=6 per group). One group of allograft recipients was treated with leflunomide (10 mg/kg/24 hr/orally) and cyclosporine (5 mg/kg24 hr/orally) starting 2 days before to surgery. Treatment continued for 60 days or until graft rejection. Untreated allografts were rejected over 6-8 days. After isograft transplantation, weight bearing began by day 17-25 after surgery. Sensory function was restored by 50 days after surgery. All allografts in the drug-treated group survived the 60-day period; survival in this group was significantly longer (P=0.0001) than the untreated controls. Weight bearing began by day 30, but was incomplete in two rats at 60 days. Peroneal nerve function was present in half the rats at 60 days after surgery. Leflunomide combined with cyclosporine prevented whole limb allograft rejection across a major histocompatibility barrier.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

SummaryEight conditioned research dogs, whose body weight was between 25 and 30 kg, had unilateral implantation of a Leeds-Keio (LK) synthetic implant (Dacron polyester) immediately after severance and removal of the cranial cruciate ligament (CCL). A rectangular- shaped autograft, composed of fascia lata, was placed within the LK implant in four of the eight dogs; the other four dogs received the LK implant only. Modified Robert Jones bandages were placed on the limbs that had been operated upon; the bandages were left in place for 48 hours. After that time the limbs were not splinted. The dogs were confined to 1.5 m X 3 m kennels, with twice daily leash walks for the duration of the study. Although the joints that received the LK implants were stable immediately after the operation, they all developed progressive effusion, crepitation and instability accompanied by clinically apparent lameness. All of the dogs were euthanatized three months after the operations. Gross examination of the eight joints that had been operated upon confirmed the presence of degenerative joint disease. Three of the eight dogs had “bucket handle” type tears of their medial meniscus in the joints with the LK implant, and the medial menisci in the other five stifle joints that received LK implants had a fibrillated surface texture. Five of the eight LK implants had either ruptured completely or were stretched and frayed to the point where mechanical testing was not justified. Mean breaking strength of the 3 intact LK implants was 6.6% of the contralateral, control CCL. Ingrowth of fibrous tissue into the LK implants was minimal. The synovium from all joints with LK implants had moderate to severely increased cellularity within the synovial villi and supporting stroma consisting predominantly of lymphocytes, plasma cells and macrophages. Multinucleated giant cells formed aggregates around irregular shaped strands of particulate material which was translucent and refractive. Under polarized light, these particles were bright yellow to green consistent with Dacron particles. It was concluded that the LK implant, as implanted in this study, was not a satisfactory replacement for the CCL in dogs.The Leeds-Keio synthetic replacement for the canine cranial cruciate ligament was implanted in eight dogs. All eight dogs developed progressive instability and secondary degenerative joint disease over a period of three months after the operation. It was concluded that the Leeds-Keio ligament, as implanted in this study, is not satisfactory for use in dogs.

To evaluate differences in contrast uptake in normal and cancerous lymph nodes on indirect computed tomography (CT) in swine, we conducted lymphographic examinations after subcutaneous injection of a lymphotropic iodinated nanoparticle suspension.Perilesional subcutaneous contrast injections (2 ml per lesion) of a 15% wt/vol iodinated nanoparticle suspension were made in immature Sinclair miniature swine (n = 5) with cutaneous melanomas. Average attenuation, iodine concentration, node volume, and total iodine uptake were estimated on the CT scans for each opacified lymph node 24 hr after injection. Nodes were classified as normal or cancerous microscopically, and the percentage of tumor replacement was estimated in cancerous nodes.Average attenuation and iodine concentration were higher in normal nodes, and total iodine uptake was higher in cancerous nodes with greater than 25% replacement (p.05). Architectural alterations in opacified cancerous nodes included medullary filling defects, expansile cortical lesions, and disruption of corticomedullary junctions.Quantitative and qualitative differences in iodinated nanoparticle enhancement characteristics are useful in distinguishing between normal and cancerous lymph nodes on indirect CT lymphography examinations.

Successful transplantation of cryopreserved complex tissues has not yet been reported. We wanted to test whether microvascular transplantation of cryopreserved knee joints can be achieved in rats. Twenty-five knee joints were cryopreserved and then microsurgically transplanted to the groin of inbred recipient animals. Ten fresh knee joints were transplanted as controls. One week after transplantation, the transplants were evaluated in situ for pedicle patency and tissue perfusion and then harvested for histology. In all surviving rats (n = 21), the cryopreserved knee joints were well perfused immediately after transplantation. At harvest, 14 knee joints appeared viable with a patent pedicle, whereas 6 were questionably viable and demonstrated arterial thrombosis. Histologically, the viable joints revealed an intact bone and cartilage matrix; however, the number of viable chondrocytes and osteocytes was reduced. Additionally, endothelial cell plugs in some areas of the capillary bed led to scattered microinfarctions despite a patent vascular pedicle. The nonperfused transplants showed infarction. In conclusion, we have demonstrated successful microvascular transplantation of cryopreserved knee joints with reduced short-term viability. Long-term studies must determine whether full recovery occurs.

Rationale and Objectives. We evaluated the imaging characteristics of an iodinated particulate contrast agent for indirect computed tomography (CT) lymphography of normal subdiaphragmatic lymph nodes in dogs. Methods. Four milliliters of a 15% (wt/vol) iodinated nanoparticle suspension was injected into the gastric, colonic, rectal, or cervical submucosa, loose paraprostatic fascia, or metatarsal subcutaneous tissues in 10 healthy beagles. Endoscopic, CT, or ultrasound guidance was used when necessary to facilitate contrast agent delivery. CT and radiographic images were obtained prior to contrast administration and at 4 hr, 24 hr, and 7 days postcontrast injection. Postmortem examinations were then conducted. Results. CT images showed enhancement of regional lymph nodes draining the various injection sites. The mean attenuation of opacified nodes was 678 ± 463 Hounsfield units 24 hr after injection and remained elevated 7 days later. Lymph node opacification on CT images correlated well with the node location observed on postmortem examinations. Conclusion. Subdiaphragmatic lymph nodes can be effectively opacified using an iodinated nanoparticle contrast agent for indirect CT lymphography.

OBJECTIVE We evaluated the effect of particle size on MR contrast-enhancing properties of arabinogalactan-coated superparamagnetic iron oxide (AG-SPIO) in tumor-bearing rats. MATERIALS AND METHODS T2*-weighted gradient-recalled echo MR studies were performed on rats with surgically implanted liver tumors before and after AG-SPIO administration. Contrast-to-noise ratio (CNR) and percent contrast enhancement (PCE) were calculated for animals given small (15.7 +/- 9.5 nm: n = 6), medium (49.1 +/- 19.7 nm; n = 4), and large (86.9 +/- 27.5 nm; n = 4) particles intravenously (10 mumol Fe/kg). RESULTS Postcontrast CNRs were 15.8 +/- 6.9, 8.9 +/- 4.1, and 10.0 +/- 1.8 for small, medium, and large particle groups, respectively. The PCE was -60.0 +/- 3.3, -75.5 +/- 7.9, and -80.5 +/- 1.2%. There was a significant difference in preversus postcontrast CNR for all particle sizes (p < 0.001) and in PCE for small particles as compared with the two larger sizes (p < 0.001). There was no between-group statistical difference in postcontrast CNR for any particle size. CONCLUSION Larger AG-SPIO particles slightly improve liver contrast enhancement, but have no significant effect on hepatic lesion detection as assessed by CNR.

Rationale and Objectives. We investigated the feasibility of percutaneous radiofrequency (RF) electrocautery in ablation of prostate tissue in dogs. Methods. We used six dogs in whom a specially designed needle was placed percutaneously into the prostate. RF electrocautery was applied to the needle and treatment was monitored with ultrasound. Animals were sacrificed and gross examination of the prostate and surrounding tissues was performed. Histopathologic examinations of the prostate were also performed. Results. The treatment zone appeared as an elliptical echogenic focus on ultrasound that increased in size with the application of current. Gross and histopathologic correlation demonstrated that the treatment area included a central area of char with a surrounding area of coagulation. There were no deleterious effects to surrounding tissues. Conclusion. Our results demonstrate the feasibility of percutaneous ultrasound-guided RF electrocautery ablation of canine prostate tissue.

Purpose: In this study, we examine the immunomodulatory effects and antitumor activity of tamoxifen and letrozole when combined with the human epithelial mucin (hMUC1)-specific vaccine, L-BLP25, in the hMUC1-expressing mammary tumor (MMT) mouse model. Experimental Design: Dose-finding studies were conducted for both tamoxifen and letrozole. Letrozole and L-BLP25 combination studies used 69 MMT female mice assigned to five groups: untreated, cyclophosphamide + placebo, cyclophosphamide + L-BLP25, letrozole 0.8 mg/kg, and cyclophosphamide + L-BLP25 + letrozole. Tamoxifen and L-BLP25 combination studies used 48 MMT female mice assigned to five treatment groups: untreated, cyclophosphamide + placebo, cyclophosphamide + L-BLP25, tamoxifen 50 mg/kg, and cyclophosphamide + L-BLP25 + tamoxifen 50 mg/kg group. Mice were injected subcutaneously with L-BLP25 (10 μg) weekly for 8 weeks. Serum cytokines were serially measured using a Luminex assay, whereas splenocytes at termination were analyzed by ELISpot to determine T-helper (TH)1/TH2 polarization of immune response. Results: Daily oral doses of 50 and 0.8 mg/kg of tamoxifen and letrozole, respectively, resulted in a significant survival advantage over controls (P < 0.05). A predominant TH1-polarized immune response in vaccinated mice was seen with or without tamoxifen or letrozole treatments. In the L-BLP25 plus letrozole treatment group, statistically significant (P < 0.05) additive antitumor activity was observed, whereas tamoxifen plus L-BLP25 was not significantly different (P > 0.05). Conclusion: The results of this study show that hormonal therapy does not interfere with L-BLP25–induced predominant TH1 response, and the combination of L-BLP25 with letrozole has additive antitumor activity in the MMT mouse model. Clin Cancer Res; 18(10); 2861–71. ©2012 AACR.

Rationale and Objectives. We evaluated the imaging characteristics of an interstitially or intraperitoneally delivered iodinated particulate contrast agent for computed tomography (CT) lymphography of the craniocervical and thoracic lymph nodes. Methods. We injected 2–4 ml of 15% wt/vol iodinated nanoparticle suspension subcutaneously, submucosally, or intraperitoneally in eight normal dogs. CT and plain radiographic images were obtained prior to contrast administration and 4 hr, 24 hr, and 7 days after injection. Correlation was made to detailed postmortem assessment. Results. CT images showed enhancement of regional nodes draining injection sites. Mean attenuation of opacified nodes was 313 ± 297 (mean ± standard deviation), 536 ± 453, and 492 ± 372 Hounsfield units at 4 hr, 24 hr, and 7 days postinjection, respectively. Lymph node opacification on CT images correlated well with node location found at postmortem. Conclusion. Craniocervical and thoracic lymph nodes can be effectively opacified from interstitial or intraperitoneal delivery of this iodinated nanoparticulate contrast agent.

OBJECTIVE Ospemifene, a new drug indicated for the treatment of vulvovaginal atrophy, has completed phase III clinical trials. A condition affecting millions of women worldwide, vulvovaginal atrophy has long been treated with estrogen therapy. Estrogen treatment carries with it risks of thromboembolism, endometrial proliferative effects, and breast cancer promotion. In this study, we test the effects of three dosing levels of ospemifene in both the prevention and treatment of breast cancer in the MTag.Tg mouse model. METHODS The polyomavirus middle-T transgenic mouse model (MTag.Tg), which produces synchronized, multifocal mammary tumors in the immunologically intact C57BL/6 background, was used to examine the impact of ospemifene treatment. First, a cell line derived from an MTag.Tg mouse tumor (MTag 34) was treated in vitro with ospemifene and its major metabolite, 4-hydroxyospemifene (4-OH ospemifene). MTag.Tg mice were treated daily by gavage with three different doses of ospemifene (5, 25, and 50 mg/kg) before or after the development of mammary tumors. Survival and tumor development results were used to determine the effect of ospemifene treatment on mammary tumors in both the preventive and treatment settings. RESULTS Tumors and the MTag 34 cell line were positive for estrogen receptor expression. The MTag 34 line was not stimulated by ospemifene or its major, active metabolite 4-OH ospemifene in vitro. Ospemifene increased survival time and exerted an antitumor effect on the development and growth of estrogen receptor-positive mammary tumors in the MTag.Tg mouse model at the 50-mg/kg dose. The levels of ospemifene and 4-OH ospemifene in both the tumors and plasma of mice confirmed the dosing. Ospemifene did not exert an estrogenic effect in the breast tissue at doses equivalent to human dosing. CONCLUSIONS Ospemifene prevents and treats estrogen receptor-positive MTag.Tg mammary tumors in this immune-intact mouse model in a dose-dependent fashion. Ospemifene drug levels in the plasma of treated mice were comparable with those found in humans. Combined with our previous data, ospemifene does not seem to pose a breast cancer risk in animals and slows down cancer development and progression in the MTag.Tg model.

Here we investigated a pharmacological approach to inhibit spermatogenesis in the mouse model by manipulating retinoid signaling using low doses of the pan-retinoic acid receptor (RAR) antagonist BMS-189453. Spermatogenesis was disrupted, with a failure of spermatid alignment and sperm release and loss of germ cells into lumen, abnormalities that resembled those in vitamin A-deficient and RARα-knockout testes. Importantly, the induced sterility was reversible. Enhanced efficacy and a lengthened infertility period with full recovery of spermatogenesis were observed using systematically modified dosing regimens. Hematology, serum chemistry, and hormonal and pathological evaluations revealed no detectable abnormalities or adverse side effects except the distinct testicular pathology. Our results suggest that testes are exquisitely sensitive to disruption of retinoid signaling and that RAR antagonists may represent new lead molecules in developing nonsteroidal male contraceptives.

Two hyperplastic parathyroid glands and three solitary parathyroid adenomas were identified using high-resolution ultrasonography in five adult dogs with persistent hypercalcemia. Ultrasonographic features of parathyroid adenomas included visualization of a round or oval, 5 mm or larger, hypoechoic mass in the cranial pole of one thyroid lobe. Each mass had well-defined margins between the thyroid gland and parathyroid adenoma, reduced echogenicity of the adenoma compared to surrounding thyroid parenchyma, and distal enhancement. Ultrasonographic features of the hyperplastic parathyroid glands included hypoechogenicity, compared to surrounding thyroid parenchyma, and a size of approximately 2 mm. Hyperplastic parathyroids were well marginated in one dog and poorly marginated in another dog.

The boxer breed of dog is at high risk for a variety of neoplasms including lymphoma. In this observational study, tissue sections from boxer dogs with lymphoma were immunostained for T and B lymphocyte distinction, and the results compared with similar studies carried out on lymphoma tissues from temporally selected cohorts of golden retriever and rottweiler dogs. The frequency of T-cell lymphomas was significantly (P < 0.001 for all comparisons) higher in the boxers than in the rottweilers or golden retrievers. We are unaware of other reports linking immunotype of canine lymphoma with breed; whether other brachycephalic breeds of dogs have a similar preponderance of T-cell lymphoma awaits further study.

Objectives Low-power multipulse contrast ultrasound imaging provides a promising tool to quantify angiogenesis noninvasively when used with contrast agents targeted to vascular markers expressed by the angiogenic endothelium. Targeted ultrasound contrast agents, with a diameter on the order of micrometers, cannot extravasate and therefore are targeted solely to receptors expressed by the vascular endothelium. The aim of this study was to evaluate the potential of a low-power multipulse imaging sequence, CadenceTM contrast pulse sequencing (CPS), combined with targeted contrast agents to quantify angiogenesis. Material and Methods Targeted microbubbles were prepared by conjugating echistatin via biotin–avidin linkage to the surface of a phospholipid microbubble shell. The density of echistatin present on the shell was confirmed with flow-cytometry and quantified by total fluorescence. The binding of targeted microbubbles was evaluated in vitro by quantifying the adherence of targeted microbubbles to rat aortic endothelial cells, compared with control (nontargeted) microbubbles. The circulation time and adherence of targeted microbubbles was evaluated in vivo in a Matrigel model in rats and compared with control microbubbles using CPS in addition to a destructive ultrasound pulse. Results Using only the low-power CPS pulse, the echo intensity produced in the neovasculature of the Matrigel pellet was significantly greater with targeted microbubbles than with the control contrast agent (p

Objective— To determine the relative importance of ischemic injury to delayed graft function (DGF) in cats. Study Design— Experimental study. Animals— Six intact female cats. Methods— Cats had renal autograft transplantation without ureteral transection and reimplantation and a contralateral nephrectomy. Serum creatinine and blood urea nitrogen (BUN) concentrations were measured regularly and abdominal ultrasound was performed before surgery, the day after surgery and twice weekly thereafter. Ultrasound-guided renal biopsy was performed on day 7. Cats were euthanatized on day 21. Histology of the autograft, ureter, bladder, vascular anastomoses sites, and contralateral kidney were performed. Observations were compared with those from an historic group of research cats that had extravesicular ureteroneocystostomy and contralateral nephrectomy.1 Results— Five cats completed the study. Serum creatinine and BUN concentrations increased after surgery, peaking at 3.2±0.8 and 77.6±15.9 mg/dL, respectively, 1–2 days after surgery. Serum creatinine concentration returned to the reference interval by 6 days after surgery. BUN gradually decreased in all cats but did not return to the reference interval by study end. Serum creatinine and BUN concentrations were consistently lower but not significantly so (P=.29 and .56, respectively) compared with the historic ureteroneocystostomy group. No ultrasonographic abnormalities or renal biopsy histologic abnormalities were observed. At necropsy, 1 autograft had generalized interstitial fibrosis. Conclusion— Harvesting the renal graft and the ischemia before revascularization causes impaired renal function after engraftment. Clinical Relevance— The process of harvesting and reimplanting the renal graft can contribute to DGF in cats, independent of ureteral obstruction.

Local ablative therapy of breast cancer represents the next frontier in the evolution of minimally-invasive breast conservation therapy. We performed this Phase II trial to determine the efficacy and safety of Radiofrequency (RF) ablation of small invasive breast carcinomas. Seventeen patients with biopsy-proven invasive breast cancer, ≤ 1.5 cm in diameter were enrolled in this trial. Under ultrasound guidance, the tumor and a 5 mm margin of surrounding breast tissue were ablated with saline-cooled RF electrode followed by surgical resection. Pathologic and immunohistochemical stains were performed to assess tumor viability. We examined whether loss of ER, PR receptor and pancytokeratin expression following RF ablation would correlate with non-viability. Fifteen patients completed the treatment. The mean tumor size was 1.28 cm. The mean ablation time was 21 minutes using a mean power of 35.5 watts. During ablation, the tumors became progressively echogenic that corresponded with the region of severe electrocautery injury at pathological examination. Of the 15 treated patients, NADPH viability staining was available for 14 patients and in 13 (92.8%), there was no evidence of viable malignant cells. ER, PR expression and pancytokeratin immunohistochemistry analysis were unreliable surrogates for determining non-viability. Following RF ablation, 2 patients developed skin puckering. RF ablation is a promising minimally invasive treatment of small breast carcinomas, as it can achieve effective cell killing with a low complication rate. Further research is necessary to optimize this image-guided technique and evaluate its future role as the sole local therapy.

The immunosuppressive effects of leflunomide and cyclosporine were evaluated in a rat neurovascularized myocutaneous allograft model. Inbred Brown-Norway and Lewis rats were served as donors and recipients, respectively. All recipients were observed for 60 days or until allograft rejection occurred. All isograft controls (Lewis to Lewis, n=6) survived uneventfully. All control allografts (n=6) were rejected within 6 days. Allograft recipients (n=6) administered leflunomide (10 mg/kg/24 hr) rejected their allografts in 28.50+/-6.12 days, and allograft recipients (n=6), administered cyclosporine (5 mg/kg/24 hr) rejected their allografts in 24.33+/-10.48 days. When allograft recipients were administered a combination of leflunomide and cyclosporine (10 mg/kg/24 hr and 5 mg/kg/24 hr, respectively), all allografts survived to 60 days with only partial rejection of the skin of one graft. The neuromuscular function of the allografts of the rats receiving combination therapy was comparable to that of the isografts. The combination of leflunomide and cyclosporine controlled myocutaneous allorejection despite a strong immunological challenge.

Background ISATX247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first study to compare the survival times of renal allografts in nonhuman primates treated with either ISATX247 or cyclosporine. Methods Adult, male cynomolgus monkeys were divided into blood-group compatible and mixed-lymphocyte, stimulation-mismatched, donor-recipient pairs. Heterotopic renal transplantation and bilateral native nephrectomies were performed. The monkeys were placed into either an ISATX247 or cyclosporine treatment group. Both groups were dosed twice daily to maintain a 12-hour drug-trough level of 150 ng/mL. Whole-blood concentrations of ISATX247 and cyclosporine, complete blood counts, and serum chemistry profiles were performed three times a week. Euthanasia was performed if the serum creatinine concentration became 7 or more mg/dL or a serious complication developed. Results The group receiving ISATX247 (n=8) survived significantly (P=0.0036) longer than the group receiving cyclosporine (n=7). The mean trough blood concentration of ISATX247 was 120 +/- 32 ng/mL and cyclosporine was 189 +/- 130 ng/mL. The average area under the curve 0-12 for ISATX247 was 6045 +/- 1679 ng/mL/hr and for cyclosporine was 4919 +/- 823 ng/mL/hr. The average calcineurin inhibition at trough blood concentrations was 80 +/- 11% for ISATX247 and 48 +/- 12% for cyclosporine. Conclusions Allografts in monkeys treated with ISATX247 survived significantly longer than those treated with cyclosporine. On the basis of survival times and degree of calcineurin inhibition, ISATX247 is a more potent immunosuppressive agent than cyclosporine in this nonhuman primate model of renal-allograft transplantation.

Background. Occult systemic inflammation, as manifested by increased levels of C-reactive protein (CRP), identify patients at increased risk for renal allograft rejection. The mechanisms linking occult systemic inflammation to these adverse outcomes remain unclear. The purpose of this study was to examine the anatomic and physiologic effects of occult pretransplantation systemic inflammation on posttransplantation allograft outcome in a nonhuman primate model. Methods. Seventy-one healthy male Rhesus macaques were stratified into quartiles based on serum CRP. Five high quartile and six low quartile animals underwent common iliac artery transplantation from male donors. Duplex ultrasound measured graft flow at 3 weeks postoperatively; luminal narrowing was assessed by graft/femoral peak systolic velocity ratio. At 6 weeks, the grafts were harvested and morphometry studies were performed. Vessel wall changes were assessed by measuring the intimal medial area. Results. The allografts placed in high CRP quartile animals had more luminal narrowing by 3 weeks than those placed in low quartile animals, as evidenced by a higher mean graft/femoral peak systolic velocity ratio (1.6 vs. 0.90, P=0.006). Morphometry studies after graft harvest showed increased vessel wall area in the high quartile group versus the low quartile group (1.39 mm2 vs. 1.03 mm2, P=0.018). Conclusions. Occult pretransplantation systemic inflammation is associated with increased intimal thickening and stenosis after arterial allograft transplantation in a primate model. Additional studies are needed to confirm these results and to further investigate potential mechanisms linking pretransplantation systemic inflammation to adverse outcomes after transplantation.