Contrast Enhanced Computed Tomography Findings in 105 Horse Distal Extremities.

The poor soft tissue conspicuity of CT can be improved by using intra-arterial CT Angiography (CTA), and intra-articular and intra-bursal contrast enhanced CT (CTAR). This retrospective study describes a combination protocol of CT and CTA of the horse's foot, and CTAR of the distal interphalangeal joint and navicular bursa. It is hypothesized this would provide a comprehensive overview of the range and severity of distal limb pathology. Radiology reports of all horses admitted for distal limb CT over a 5 year period were reviewed. All horses with a complete four stage CT examination and radiology report with lameness isolated to the foot were included. Twenty seven imaging findings using a four grade semiquantitative severity scoring system contributing towards six main diagnostic categories were described. One hundred and five examinations on 56 horses revealed a diagnosis of navicular bone disease in 64%, deep digital flexor tendinopathy in 43%, distal interphalangeal osteoarthritis in 35%, navicular bursitis in 31%, distal interphalangeal collateral ligament desmopathy in 26%, and hoof capsule and distal phalanx pathology in 10%. Only 25% of the navicular bone disease cases were considered clinically significant. The majority of deep digital flexor tendon lesions (77%) and distal interphalangeal joint osteoarthritis (51%) were considered significant. Approximately one third of navicular bursa (37%) and collateral ligament (33%) abnormalities were considered significant. Navicular bursa abnormalities were associated with navicular bone and deep digital flexor tendon lesions. The findings support the hypothesis and the use of this protocol for evaluation of foot lameness.

Investigation of percentage changes in pulse wave transit time induced by mini-fluid challenges to predict fluid responsiveness in ventilated dogs.

OBJECTIVE: To investigate whether percentage changes in pulse wave transit time (PWTT%Δ) induced by mini-fluid challenges predict fluid responsiveness in mechanically ventilated anesthetized dogs. DESIGN: Prospective experimental trial. SETTING: University teaching hospital. ANIMALS: Twelve Harrier hounds. INTERVENTION: Each dog was anesthetized with propofol and isoflurane after premedication with acepromazine, mechanically ventilated, and had a fluid challenge. This was repeated 4 weeks later. The fluid challenge, 10 mL/kg of colloid administration over 13 minutes, consisted of 3 intermittent mini-fluid challenges (1 mL/kg of each over a minute) with a minute interval, and the remaining colloid administration (7 mL/kg) over 7 minutes. MEASUREMENTS AND MAIN RESULTS: Percentage change in velocity time integral of pulmonary arterial flow by echocardiography was calculated as an indication of change in stroke volume. Fluid responsiveness was defined as percentage change in velocity time integral ≥ 15% after 10 mL/kg colloid. Dogs responded on 14 fluid challenges and did not on 10. After 1, 2, 3, and 10 mL/kg of fluid challenge, PWTT%Δ1, 2, 3, 10 were measured. Receiver operator characteristic (ROC) curves were generated and areas under ROC curve were calculated for PWTT%Δ1, 2, 3 . A gray zone approach was used to identify the clinically inconclusive range. The area under the ROC curve for PWTT%Δ3 was 0.91 (P = 0.001). Cutoff value for PWTT%Δ3 was -2.5% (sensitivity: 86%, specificity: 90%). The gray zone for PWTT%Δ3 was identified as between -2.9% to -1.9% for which fluid responsiveness could not be predicted reliably in 6 out of 24 fluid challenges. CONCLUSIONS: In mechanically ventilated anesthetized dogs given a mini-fluid challenge of 3 mL/kg of colloid, PWTT%Δ could predict fluid responsiveness although the gray zone should be considered.

Obstructive sleep apnea in a Chihuahua successfully managed with ondansetron.

While the persistence of clinical signs related to brachycephalic obstructive airway syndrome, particularly sleep-disordered breathing patterns following appropriate surgical management is likely to be relatively rare, this potential sequela needs to be considered, along with being aware of possible medical management options such as serotonin antagonists.

Dermoid sinus type VI associated with spina bifida and tethered cord syndrome in a French Bulldog.

A 4-mo-old French bulldog was presented with acute onset pain and reluctance to move. A tubular structure arising in the dorsal thoracic midline and extending from a cutaneous orifice into deeper tissues was palpated on physical examination. Computed tomography with sinography revealed a dermoid sinus associated with spina bifida at the level of T3-T4. On surgical exploration, the dermoid sinus was found to communicate with the dura. Histology confirmed the diagnosis and classification as a type VI dermoid sinus. The pain response and hyperesthesia were suspected to be the result of tethered cord syndrome. Complete resolution of clinical signs was appreciated post-surgery, with the patient still free of clinical signs 3 mo later.

Evaluation of pulse pressure variation and pleth variability index to predict fluid responsiveness in mechanically ventilated isoflurane-anesthetized dogs.

OBJECTIVE: To evaluate whether pulse pressure variation (PPV) and pleth variability index (PVI) are more accurate than central venous pressure (CVP) for predicting fluid responsiveness in mechanically ventilated isoflurane-anesthetized dogs after premedication with acepromazine. DESIGN: Prospective experimental trial. SETTING: University teaching hospital. ANIMALS: Twelve Harrier hound dogs. INTERVENTIONS: Each dog was anesthetized and had a fluid challenge performed. This was repeated 4 weeks later for a total of 24 fluid challenges. After premedication with intramuscular acepromazine, anesthesia was induced with propofol and maintained with isoflurane. The dogs were mechanically ventilated with constant settings. The fluid challenge consisted of 10 mL/kg of 6% hydroxyethyl starch intravenously over 13 minutes. MEASUREMENTS AND MAIN RESULTS: Before and after the fluid challenge, PPV, PVI, CVP, and other hemodynamics were recorded. Change in velocity time integral of pulmonary arterial blood flow by echocardiography was calculated as an indication of change in stroke volume. A fluid responder was defined as an increase in velocity time integral ≥ 15%. Receiver operator characteristic (ROC) curves were used to determine cutoff values. Areas under ROC curve were calculated and compared. Dogs responded on 14 fluid challenges and did not on 10. Cutoff values for PPV and PVI were 11% (sensitivity 79%; specificity 80%) and 9.3% (sensitivity 86%; specificity 70%), respectively. The areas under the ROC curve of PPV [0.85, 95% confidence interval (CI): 0.70-1.00, P = 0.038] and PVI (0.84, 95% CI: 0.68-1.00, P = 0.043) were significantly higher than CVP (0.56, 95% CI: 0.32-0.81). CONCLUSIONS: PPV and PVI predicted fluid responsiveness more accurately than CVP and may be useful to guide fluid administration in mechanically ventilated isoflurane-anesthetized dogs after premedication with acepromazine.

Emergence of nasal chondrosarcoma in a dog with nasal polyposis.

Whilst the malignant transformation of nasal polyps or secondary development of nasal neoplasia after chronic inflammation is likely to be relatively rare, this potential complication should be considered, and the clinician should be vigilant for evidence of malignant transformation.

RADIOGRAPHIC MEASUREMENTS OF HOOF BALANCE ARE SIGNIFICANTLY INFLUENCED BY A HORSE'S STANCE.

Hoof balance radiographs are commonly used as the basis for corrective farriery decision-making in horses, however there are limited published data quantifying effects of the stance of the horse or the horizontal radiographic beam angle. In this analytical study, the influence of variation of the horse's stance in the craniocaudal and lateromodial plane on hoof balance measurements as well as the influence of variation of the horizontal radiographic beam angle on dorsopalmar hoof balance measurements was examined. Distal left thoracic limb lateromedial radiographs were acquired using a standardized protocol while varying the craniocaudal stance of five horses, each selected to be sound and conformationally normal. Dorsopalmar foot radiographs were acquired while varying the lateromedial stance; and variable angle horizontal beam dorsopalmar foot radiographs were acquired while keeping the limb position constant. Analyses of measurements demonstrated that hoof pastern angle had a linear relationship (R2 = 0.89, P < 0.001) with craniocaudal stance of the horse. The relationship of joint angle and stance was greater for the distal interphalangeal joint angle (R2 = 0.89, P < 0.001) than the proximal interphalangeal joint angle (R2 = 0.65, P = 0.001). The distal phalanx angle did not change with craniocaudal stance variation. The proximal interphalangeal joint width, distal interphalangeal joint width, or distal phalanx height did not change with lateromedial stance variation, nor within a 15 degree dorsolateral to caudomedial and dorsomedial to caudolateral variation from the dorsopalmar axis. Findings indicated that positioning of the thoracic limb needs to be considered during radiographic interpretation and decision-making for corrective farriery.

An imaging investigation of in situ uroliths in hospitalized cats in New Zealand and in the United States.

The submission rates of feline uroliths to laboratories and the composition of uroliths have been reported in studies. The prevalence of uroliths reported on imaging findings has not been published. The objective of this retrospective study was to use imaging data to investigate the anatomical location and the prevalence of macroscopic in situ uroliths in cats. Radiographs, sonograms and imaging reports from two cohorts of cats (from New Zealand (n = 497) and the United States (n = 693)) from 2004-2013 were reviewed for the presence of in situ uroliths. Uroliths were categorized by their location in the lower or upper urinary tract. Radiographic studies were performed on 43% (212/497) of the cats from New Zealand and 50% (349/693) of the cats from the USA. Sonographic studies were performed on 57% (285/497) of the cats from New Zealand and 50% (344/693) of the cats from the USA. The total prevalence of uroliths was 3% in the New Zealand cohort and 13% in the USA cohort. Lower tract urolith prevalence in the New Zealand cohort was 2.4% (5/212) in cats ≤ 6y and 1.1% (3/285) in cats >6y. Upper tract urolith prevalence in the New Zealand cohort was 0.5% (1/212) in cats ≤ 6y and 1.8% (5/285) in cats >6y. Lower tract urolith prevalence in the United States cohort was 6.0% (11/183) in cats ≤ 6y and 2.9% (15/510) in cats >6y. Upper tract urolith prevalence in the United States cohort was 2.7% (5/183) in cats ≤ 6y and 10.2% (52/510) in cats >6y. The prevalence of uroliths in the upper tract or lower tract was low in the New Zealand cohort compared to that of cats in the USA cohort, irrespective of age category. Geographical location may be important when evaluating risk factors for feline urolithiasis.

IMAGING DIAGNOSIS--USE OF RADIOGRAPHY AND COMPUTED TOMOGRAPHY IN THE DIAGNOSIS OF A MINERALIZED YOLK SAC IN A BROWN KIWI (APTERYX MANTELLI).

A 12-day-old Brown Kiwi (Apteryx mantelli) was presented with anorexia, torticollis, head-tilt, and coelomic distension. Radiographs showed an ill-defined, fat-opaque, coelomic mass displacing viscera craniodorsally. Curvilinear mineral opacities were superimposed over the ventral aspect of the mass. Computed tomography demonstrated the presence of mineral within the periphery of a fat attenuating mass consistent with a retained yolk sac. A deutectomy (yolk sac excision) was performed. Histopathology of the excised tissue confirmed the diagnosis of a retained yolk sac with multifocal mineralization.

IgG2 antibodies against a clinical grade Plasmodium falciparum CSP vaccine antigen associate with protection against transgenic sporozoite challenge in mice.

The availability of a highly purified and well characterized circumsporozoite protein (CSP) is essential to improve upon the partial success of recombinant CSP-based malaria vaccine candidates. Soluble, near full-length, Plasmodium falciparum CSP vaccine antigen (CS/D) was produced in E. coli under bio-production conditions that comply with current Good Manufacturing Practices (cGMP). A mouse immunogenicity study was conducted using a stable oil-in-water emulsion (SE) of CS/D in combination with the Toll-Like Receptor 4 (TLR4) agonist Glucopyranosyl Lipid A (GLA/SE), or one of two TLR7/8 agonists: R848 (un-conjugated) or 3M-051 (covalently conjugated). Compared to Alum and SE, GLA/SE induced higher CS/D specific antibody response in Balb/c mice. Subclass analysis showed higher IgG2:IgG1 ratio of GLA/SE induced antibodies as compared to Alum and SE. TLR synergy was not observed when soluble R848 was mixed with GLA/SE. Antibody response of 3M051 formulations in Balb/c was similar to GLA/SE, except for the higher IgG2:IgG1 ratio and a trend towards higher T cell responses in 3M051 containing groups. However, no synergistic enhancement of antibody and T cell response was evident when 3M051 conjugate was mixed with GLA/SE. In C57Bl/6 mice, CS/D adjuvanted with 3M051/SE or GLA/SE induced higher CSP repeat specific titers compared to SE. While, 3M051 induced antibodies had high IgG2c:IgG1 ratio, GLA/SE promoted high levels of both IgG1 and IgG2c. GLA/SE also induced more potent T-cell responses compared to SE in two independent C57/BL6 vaccination studies, suggesting a balanced and productive T(H1)/T(H2) response. GLA and 3M-051 similarly enhanced the protective efficacy of CS/D against challenge with a transgenic P. berghei parasite and most importantly, high levels of cytophilic IgG2 antibodies were associated with protection in this model. Our data indicated that the cGMP-grade, soluble CS/D antigen combined with the TLR4-containing adjuvant GLA/SE warrants further evaluation for protective responses in humans.

Vaccine adjuvant activity of 3M-052: an imidazoquinoline designed for local activity without systemic cytokine induction.

The human Toll-like receptors (TLRs) are a family of receptors, which sense the presence of various structural elements of pathogens and damaged or effete components in the host. As they do so, they activate two critical arms of host defense, the rapid innate immune response and an adaptive immune response. The innate immune response is typified by the generation of Th1 cytokines, chemokines and type 1 interferons. As such, agonists for the TLRs have potential as antiviral and anticancer therapeutics. They are also well suited to function as vaccine adjuvants. 3M imidazoquinoline (IRM) molecules were the first synthetic small molecules identified as TLR agonists and can affect their biological activities through TLR7, TLR8, or both. The breadth of therapeutic opportunities for this family of molecules can require formulations tailored to the specific application. One consideration is specific formulations to avoid a systemic distribution of these TLR agonists and resulting cytokine storm-like effects on the host. 3M-052 is an IRM bearing a C18 lipid moiety and designed for slow dissemination from the site of application. In the present study 3M-052 has been evaluated for its in vitro TLR activity and for its efficacy as a vaccine adjuvant using a recombinant hemagglutinin from H1N1 A/Puerto Rico/8/34. Given subcutaneously, 3M-052 drives a strong Th1 response to hemagglutinin and serum neutralization of viable H1N1 A/Puerto Rico/8/34 virus in the absence of circulating TNFα or the induction of Th1 cytokines.

The covalent modification and regulation of TLR8 in HEK-293 cells stimulated with imidazoquinoline agonists.

The mammalian TLRs (Toll-like receptors) mediate the rapid initial immune response to pathogens through recognition of pathogen-associated molecular patterns. The pathogen pattern to which TLR8 responds is ssRNA (single-stranded RNA) commonly associated with ssRNA viruses. TLR8 also responds to small, purine-like molecules including the imidazoquinoline IRMs (immune-response modifiers). The IRMs include molecules that selectively activate TLR7, selectively activate TLR8 or non-selectively activate both TLR7 and TLR8. Using HEK-293 cells (human embryonic kidney cells) stably expressing an NF-kappaB (nuclear factor kappaB)/luciferase promoter-reporter system as a model system, we have examined the regulation of TLR8 using the non-selective TLR7/8 agonist, 3M-003. Using conservative tyrosine to phenylalanine site-directed mutation, we show that of the 13 tyrosine residues resident in the cytosolic domain of TLR8, only three appear to be critical to TLR8 signalling. Two of these, Tyr898 and Tyr904, reside in the Box 1 motif and the third, Tyr1048, lies in a YXXM putative p85-binding motif. TLR8 is tyrosine-phosphorylated following 3M-003 treatment and TLR8 signalling is inhibited by tyrosine kinase inhibitors. Treatment with 3M-003 results in the association of the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase) with TLR8 and this association is inhibited by tyrosine to phenylalanine mutation of either the YXXM or Box 1 motifs. As a further consequence of activation by 3M-003, TLR8 is modified to yield both higher and lower molecular mass species. These species include a monoubiquitinated form as deduced from ubiquitin peptide sequencing by HPLC/MS/MS (tandem MS).

Oligodeoxynucleotides differentially modulate activation of TLR7 and TLR8 by imidazoquinolines.

Among the 11 human TLRs, a subfamily TLR7, TLR8, and TLR9 display similarities in structure and endosomal localization. Natural agonists consisting of nucleic acids, such as ssRNA or DNA with CpG motifs, activate the innate immune cells through these TLRs. Immune response modifiers (IRMs) of imidazoquinoline class compounds 3M-001, 3M-002, and 3M-003 have been shown to activate the innate immune system via TLR7, TLR8, and TLR7/8, respectively. In looking at the effect of the agonists of the TLR7, TLR8, and TLR9 on the activation of NF-kappaB of transfected HEK cells, we discovered that some oligodeoxynucleotides (ODNs) could modulate imidazoquinoline effects in a negative or positive manner. In this study we demonstrate that poly(T) ODNs can inhibit TLR7 and enhance TLR8 signaling events involving NF-kappaB activation in HEK cells and cytokine production (IFN-alpha, TNF, and IL-12) by human primary PBMC. In contrast, TLR3 agonist poly(I:C) does not affect imidazoquinoline-induced responses. The modulation of TLR7 and TLR8 responses is independent of CpG motifs or the nature of the ODN backbone structure. Furthermore, we show that to be an effective modulator, the ODNs need to be in the cell at the same time with either of the TLR7 or TLR8 agonist. We have also demonstrated that there is a physical interaction between IRMs and ODNs. The cross-talk between ODNs, IRMs, and TLR7 and TLR8 uncovered by this study may have practical implications in the field of microbial infections, vaccination, and tumor therapy.

The functional effects of physical interactions among Toll-like receptors 7, 8, and 9.

Toll-like receptors (TLRs) TLR1, TLR2, TLR4, and TLR6 are evolutionarily conserved, highly homologous, and localized to plasma membranes of host cells and recognize pathogen-associated molecular patterns (PAMPs) derived from bacterial membranes. These receptors cooperate in a pairwise combination to elicit or inhibit the inflammatory signals in response to certain PAMPs. The other TLRs that are evolutionarily closely related and highly homologous are TLR7, TLR8, and TLR9. They are all confined to the membranes of endosomes and recognize similar molecular structures, the oligonucleotide-based PAMPs. However, the cooperative interactions among these receptors that may modulate the inflammatory signaling in response to their cognate agonists are not reported. We report here for the first time the functional effects of one TLR on the other among TLR7, TLR8, and TLR9. The results indicate that TLR8 inhibits TLR7 and TLR9, and TLR9 inhibits TLR7 but not vice versa in HEK293 cells transfected with TLRs in a pairwise combination. This is concluded by selectively activating one TLR over the other by using small molecule TLR agonists. We also show that these inhibitory interactions are the result of direct or indirect physical interactions between the TLRs. The murine TLR8 that does not respond to any known human TLR8 agonists also inhibits both murine and human TLR7. The implications of the inhibitory interactions among these TLRs in host-pathogen recognition and subsequent inflammatory responses are not obvious. However, given the complexity in expression pattern in a particular cell type and the variation in distribution and response to different pathogens and stress signals in different cell types, the inhibitory physical interactions among these TLRs may play a role in balancing the inflammatory outcome from a given cell type to a specific challenge.

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent.

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.