Targeted Analysis of the Size Distribution of Heavy Chain-Modified Hyaluronan with Solid-State Nanopores.

The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HC•HA) complexes. While limited assessments of HC•HA have been performed previously, determining the size distribution of its HA component remains a challenge. Here, we describe a selective method for extracting HC•HA from mixtures that yields material amenable to MW analysis with a solid-state nanopore sensor. After demonstrating the approach in vitro, we validate extraction of HC•HA from osteoarthritic human synovial fluid as a model complex biological matrix. Finally, we apply our technique to pathophysiology by measuring the size distributions of HC•HA and total HA in an equine model of synovitis.

The effect of cyanoacrylate on knot elongation in three sutures used for prosthetic laryngoplasty in the horse.

OBJECTIVE: This study investigated the elongation following cyclic loading on square knots of 5 USP multifilament long-chain ultra-high molecular weight polyethylene core (UHMWPE), 2 mm woven UHMWPE tape, and 5 USP braided polyester, with and without cyanoacrylate glue. STUDY DESIGN: Experimental study. SAMPLE POPULATION: n = 4. METHODS: Three conditions (suture without knot, suture with knot, suture with knot + cyanoacrylate) were evaluated for each suture material on a mechanical test stand by measuring the increased length of the construct after cycling from 25 to 50N for 1000 repetitions at 20 mm/second. Knot elongation was determined by subtracting the length of the control suture from the suture with knot or suture with knot + cyanoacrylate. The data were analyzed with a linear regression model with robust estimation of variance. Post-hoc analysis determined the model adjusted differences (square knot vs. cyanoacrylate) as a difference from control. t-tests were conducted to identify the significant findings. RESULTS: Total elongation of polyester (6.2-7.8 mm) was greater than multifilament UHMWPE (3.4-6.4 mm) and UHMWPE tape (2-3.7 mm) for all conditions. Polyester had the lowest knot elongation (1.6 mm) and the addition of cyanoacrylate decreased knot elongation for polyester by 1 mm. CONCLUSIONS: Polyester had the most total construct elongation followed by multifilament UHMWPE and UHMWPE tape. Polyester showed the least knot elongation and cyanoacrylate decreased this knot elongation. CLINICAL SIGNIFICANCE: Total construct and knot elongation should be considered as contributing factors to loss of arytenoid abduction following prosthetic laryngoplasty when using polyester, multifilament UHMWPE, or UHMWPE tape. Addition of cyanoacrylate to polyester knots should be explored to limit elongation.

Comparison of cervical epidural morphine with intravenous morphine administration on antinociception in adult horses using thermal threshold testing.

OBJECTIVE: To compare the antinociceptive effects of morphine administered via cervical epidural catheter to intravenously administered morphine using a thermal threshold (TT) testing model in healthy adult horses. STUDY DESIGN: Prospective, randomized, blinded experimental study. ANIMALS: A total of six university-owned adult horses. METHODS: Horses were instrumented with a cervical (C1-C2) epidural catheter and TT testing device with probes at withers and thoracic limb coronary bands. All horses underwent three TT testing cycles including cervical epidural morphine administration (treatment EpiM; 0.1 mg kg-1), systemic morphine administration (treatment SystM; 0.1 mg kg-1) and no morphine administration (treatment Control). Baseline TT was established prior to treatments, and TT was tested at 15, 30, 60, 90, 120, 150, 180, 240, 300, 360, 420, 480, 600 and 720 minutes following treatment. Horses underwent a 5 day washout period between treatments and the order of treatment was randomized. Differences between treatments were analyzed with repeated measures anova. RESULTS: Systemic and epidural morphine administration resulted in significantly higher TT values compared with baseline and control treatment. The duration of effect was significantly longer in treatment EpiM (10-12 hours) than in treatment SystM (1.5-2.0 hours). Horses in treatment EpiM had significantly higher TT values at time points 180-600 minutes (withers) and 300-600 minutes (coronary band) than horses in treatment SystM. CONCLUSIONS AND CLINICAL RELEVANCE: Cervical epidural administration of morphine provided antinociceptive effects as measured by increased TT for 10-12 hours compared with 1.5-2.0 hours for intravenously administered morphine. No complications or adverse effects were noticed following epidural placement of a C1-C2 catheter and administration of morphine. The use of a cervical epidural catheter can be considered for analgesia administration in treatment of thoracic limb and cervical pain in the horse.

Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models.

BACKGROUND: Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1ß-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1ß synovitis and tarsal intra-articular lavage. RESULTS: Hyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17-20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands - 2, - 3, - 5 and - 11) as compared to lavage. CONCLUSIONS: Synovial fluid lubricin increased in response to IL-1ß synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

Inhibition of type 4 cyclic nucleotide phosphodiesterase blocks intracellular TLR signaling in chronic lymphocytic leukemia and normal hematopoietic cells.

A subset of chronic lymphocytic leukemia (CLL) BCRs interacts with Ags expressed on apoptotic cells, suggesting that CLL BCRs have the potential to internalize apoptotic cell RNA- or DNA-containing fragments with resultant activation of TLR7 or TLR9, respectively. By blocking cAMP degradation, type 4 cAMP phosphodiesterase (PDE4) inhibitors activate cAMP-mediated signaling and induce apoptosis in CLL cells. In this study, we show that autologous irradiated leukemic cells induce proliferation in CLL cells and that such proliferation is blocked by a TLR7/8/9 inhibitor, by DNase, and by the PDE4 inhibitor rolipram. Rolipram also inhibited CLL cell proliferation induced by synthetic TLR7 and TLR9 agonists, as well as TLR agonist-induced costimulatory molecule expression and TNF-a (but not IL-6 or IL-10) production. Whereas treatment with a TLR9 agonist protected IgH V region unmutated, but not mutated, CLL cells from apoptosis, PDE4 inhibitors augmented apoptosis in both subtypes, suggesting that cAMP-mediated signaling may abrogate a TLR9-mediated survival signal in prognostically unfavorable IGHV unmutated CLL cells. Rolipram inhibited both TLR7/8- and TLR9-induced IFN regulatory factor 5 and NF-kB p65 nuclear translocation. PDE4 inhibitors also blocked TLR signaling in normal human immune cells. In PBMC and CD14-positive monocytes, PDE4 inhibitors blocked IFN-a or TNF-a (but not IL-6) production, respectively, following stimulation with synthetic TLR agonists or RNA-containing immune complexes. These results suggest that PDE4 inhibitors may be of clinical utility in CLL or autoimmune diseases that are driven by TLR-mediated signaling.

IRF5 deficiency ameliorates lupus but promotes atherosclerosis and metabolic dysfunction in a mouse model of lupus-associated atherosclerosis.

Premature atherosclerosis is a severe complication of lupus and other systemic autoimmune disorders. Gain-of-function polymorphisms in IFN regulatory factor 5 (IRF5) are associated with an increased risk of developing lupus, and IRF5 deficiency in lupus mouse models ameliorates disease. However, whether IRF5 deficiency also protects against atherosclerosis development in lupus is not known. In this study, we addressed this question using the gld.apoE(-/-) mouse model. IRF5 deficiency markedly reduced lupus disease severity. Unexpectedly, despite the reduction in systemic immune activation, IRF5-deficient mice developed increased atherosclerosis and also exhibited metabolic dysregulation characterized by hyperlipidemia, increased adiposity, and insulin resistance. Levels of the atheroprotective cytokine IL-10 were reduced in aortae of IRF5-deficient mice, and in vitro studies demonstrated that IRF5 is required for IL-10 production downstream of TLR7 and TLR9 signaling in multiple immune cell types. Chimera studies showed that IRF5 deficiency in bone marrow-derived cells prevents lupus development and contributes in part to the increased atherosclerosis. Notably, IRF5 deficiency in non-bone marrow-derived cells also contributes to the increased atherosclerosis through the generation of hyperlipidemia and increased adiposity. Together, our results reveal a protective role for IRF5 in lupus-associated atherosclerosis that is mediated through the effects of IRF5 in both immune and nonimmune cells. These findings have implications for the proposed targeting of IRF5 in the treatment of autoimmune disease as global IRF5 inhibition may exacerbate cardiovascular disease in these patients.

B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile.

Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell-null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell-null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.

Connecting model species to nature: predator-induced long-term sensitization in Aplysia californica.

Previous research on sensitization in Aplysia was based entirely on unnatural noxious stimuli, usually electric shock, until our laboratory found that a natural noxious stimulus, a single sublethal lobster attack, causes short-term sensitization. We here extend that finding by demonstrating that multiple lobster attacks induce long-term sensitization (≥24 h) as well as similar, although not identical, neuronal correlates as observed after electric shock. Together these findings establish long- and short-term sensitization caused by sublethal predator attack as a natural equivalent to sensitization caused by artificial stimuli.

Phenotype and function of B cells and dendritic cells from interferon regulatory factor 5-deficient mice with and without a mutation in DOCK2.

Interferon regulatory factor 5-deficient (IRF5 (-/-) ) mice have been used for many studies of IRF5 biology. A recent report identifies a mutation in dedicator of cytokinesis 2 (DOCK2) as being responsible for the abnormal B-cell development phenotype observed in the IRF5 (-/-) line. Both dedicator of cytokinesis 2 (DOCK2) and IRF5 play important roles in immune cell function, raising the issue of whether immune effects previously associated with IRF5 are due to IRF5 or DOCK2. Here, we defined the insertion end-point of the DOCK2 mutation and designed a novel PCR to detect the mutation in genomic DNA. We confirmed the association of the DOCK2 mutation and the abnormal B-cell phenotype in our IRF5 (-/-) line and also established another IRF5 (-/-) line without the DOCK2 mutation. These two lines were used to compare the role of IRF5 in dendritic cells (DCs) and B cells in the presence or absence of the DOCK2 mutation. IRF5 deficiency reduces IFN-α, IFN-ß and IL-6 production by Toll-like receptor 9 (TLR9)- and TLR7-stimulated DCs and reduces TLR7- and TLR9-induced IL-6 production by B cells to a similar extent in the two lines. Importantly however, IRF5 (-/-) mice with the DOCK2 mutation have higher serum levels of IgG1 and lower levels of IgG2b, IgG2a/c and IgG3 than IRF5 (-/-) mice without the DOCK2 mutation, suggesting that the DOCK2 mutation confers additional Th2-type effects. Overall, these studies help clarify the function of IRF5 in B cells and DCs in the absence of the DOCK2 mutation. In addition, the PCR described will be useful for other investigators using the IRF5 (-/-) mouse line.

Dexmedetomidine preconditioning reduces ischaemia-reperfusion injury in equine model of large colon volvulus.

BACKGROUND: Large colon volvulus is a cause of colic in horses with high morbidity and mortality when not promptly treated. More treatment options are needed to improve the outcome of these cases by protecting against the damage caused by ischaemia and reperfusion injury. OBJECTIVES: To determine the effect of preconditioning with dexmedetomidine prior to induction of ischaemia-reperfusion (IR) injury in a large colon volvulus model in the horse. STUDY DESIGN: Randomised blinded in vivo experiments. METHODS: Horses received either a dexmedetomidine (DEX) or saline (CON) constant rate infusion (CRI) immediately following induction of anaesthesia. Venous, arterial, and transmural occlusion of a section of the large colon was performed for 3 h, after which the ligatures and clamps were removed to allow for reperfusion for 3 h. Biopsies of the large colon were taken at baseline, 1 and 3 h of ischaemia, and at 1 and 3 h of reperfusion. RESULTS: The severity of crypt epithelial loss (DEX = 2.1 [0.8-2.8], CON = 3.1 [2.5-4], p = 0.03) and mucosal haemorrhage was decreased (DEX = 2.1 [1.3-3], CON = 3.5 [2.5-4], p = 0.03) in group DEX compared to group CON when graded on a scale of 0-4. Crypt length remained longer (DEX = 369.5 ± 91.7 µm, CON = 238.5 ± 72.6 µm, p = 0.02) and interstitium to crypt (I:C) ratio remained lower (DEX = 1.4 (1-1.7), CON = 2.6 [1.8-5.9], p = 0.03) in group DEX compared to group CON during reperfusion. MAIN LIMITATIONS: Clinical applicability of pharmacologic preconditioning is limited. CONCLUSION: Preconditioning with a dexmedetomidine CRI prior to IR injury demonstrated a protective effect histologically on the large colon in the horse. Further investigation into postconditioning with dexmedetomidine is warranted as a possible intervention in colic cases suspected of being large colon volvulus.

Targeting biofilm infections in humans using small scale robotics.

The eradication of drug-resistant microbial biofilms remains an unresolved global health challenge. Small-scale robotics are providing innovative therapeutic and diagnostic approaches with high precision and efficacy. These approaches are rapidly moving from proof-of-concept studies to translational biomedical applications using ex vivo, animal, and clinical models. Here, we discuss the fundamental and translational aspects of how microrobots target the infection sites to disrupt the structural and functional traits of biofilms and their antimicrobial resistance mechanisms. We emphasize current approaches of mechanochemical disruption and on-site drug delivery that are supported by in vivo models and preclinical testing, while also highlighting diagnostics potential. We also discuss clinical translation challenges and provide perspectives for development of microrobotics approaches to combat biofilm infections and biofouling in humans.

Loss of effective lubricating viscosity is the primary mechanical marker of joint inflammation in equine synovitis.

Inflammation of the synovium, known as synovitis, plays an important role in the pathogenesis of osteoarthritis (OA). Synovitis involves the release of a wide variety of pro-inflammatory mediators in synovial fluid (SF) that damage the articular cartilage extracellular matrix and induce death and apoptosis in chondrocytes. The composition of synovial fluid is dramatically altered by inflammation in OA, with changes to both hyaluronic acid and lubricin, the primary lubricating molecules in SF. However, the relationship between key biochemical markers of joint inflammation and mechanical function of SF is not well understood. Here, we demonstrate the application of a novel analytical framework to measure the effective viscosity for SF lubrication of cartilage, which is distinct from conventional rheological viscosity. Notably, in a well-established equine model of synovitis, this effective lubricating viscosity decreased by up to 10,000-fold for synovitis SF compared to a ~4 fold change in conventional viscosity measurements. Further, the effective lubricating viscosity was strongly inversely correlated (r = -0.6 to -0.8) to multiple established biochemical markers of SF inflammation, including white blood cell count, prostaglandin E2 (PGE2), and chemokine ligand (CCLs) concentrations, while conventional measurements of viscosity were poorly correlated to these markers. These findings demonstrate the importance of experimental and analytical approaches to characterize functional lubricating properties of synovial fluid and their relationships to soluble biomarkers to better understand the progression of OA.

Efficacy and pharmacodynamic effect of anti-CD73 and anti-PD-L1 monoclonal antibodies in combination with cytotoxic therapy: observations from mouse tumor models.

CD73 is a cell surface 5'nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy.

The dilutional volume of morphine and xylazine administered via caudal epidural catheter affects cranial spread of analgesia in healthy standing horses.

OBJECTIVE: To test the influence of increasing injectate volumes on the regional effects of xylazine and morphine epidural analgesia, with the hypothesis that increasing volume produces more cranial spread of analgesia as determined by thermal threshold (TT) testing. ANIMALS: 6 university-owned research/teaching horses (2 mares, 4 geldings) deemed healthy on physical examination and basic lameness evaluation, aged 6-19 years and weighing 420-560 kg, were used in this prospective, randomized, blinded, cross-over experimental study. METHODS: After routine placement of a caudal epidural catheter, all animals were subsequently instrumented with a TT testing system at the withers (Location A), the cranial (Location B), and caudal (Location C) abdominal area, over the tuber coxae (Location D), and the hind limb dorsal pasterns (Location E). All horses underwent five testing cycles with 0.2 mg/kg morphine and 0.2 mg/kg xylazine diluted to 20, 35, 50, 75, and 100 mL. TT testing was performed at 2, 4, 6, 8, and 10 hours by blinded investigators. RESULTS: With increased epidural volume, significantly greater cranial spread of analgesic effect was noted. All epidural volumes caused significant changes in TT testing at location E but only the largest volume resulted in a significant TT testing change at location A. CLINICAL RELEVANCE: Volume influences the regional effects of caudal epidural analgesia in horses but might affect analgesic reliability.

IFN regulatory factor 5 is required for disease development in the FcgammaRIIB-/-Yaa and FcgammaRIIB-/- mouse models of systemic lupus erythematosus.

Polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are strongly associated in human genetic studies with an increased risk of developing the autoimmune disease systemic lupus erythematosus. However, the biological role of IRF5 in lupus pathogenesis has not previously been tested in an animal model. In this study, we show that IRF5 is absolutely required for disease development in the FcgammaRIIB(-/-)Yaa and FcgammaRIIB(-/-) lupus models. In contrast to IRF5-sufficient FcgammaRIIB(-/-)Yaa mice, IRF5-deficient FcgammaRIIB(-/-)Yaa mice do not develop lupus manifestations and have a phenotype comparable to wild-type mice. Strikingly, full expression of IRF5 is required for the development of autoimmunity, as IRF5 heterozygotes had dramatically reduced disease. One effect of IRF5 is to induce the production of the type I IFN, IFN-alpha, a cytokine implicated in lupus pathogenesis. To address the mechanism by which IRF5 promotes disease, we evaluated FcgammaRIIB(-/-)Yaa mice lacking the type I IFN receptor subunit 1. Unlike the IRF5-deficient and IRF5-heterozygous FcgammaRIIB(-/-)Yaa mice, type I IFN receptor subunit 1-deficient FcgammaRIIB(-/-)Yaa mice maintained a substantial level of residual disease. Furthermore, in FcgammaRIIB(-/-) mice lacking Yaa, IRF5-deficiency also markedly reduced disease manifestations, indicating that the beneficial effects of IRF5 deficiency in FcgammaRIIB(-/-)Yaa mice are not due only to inhibition of the enhanced TLR7 signaling associated with the Yaa mutation. Overall, we demonstrate that IRF5 plays an essential role in lupus pathogenesis in murine models and that this is mediated through pathways beyond that of type I IFN production.

Intratracheal oxygen administration increases FIO2 and PaO2 compared with intranasal administration in healthy, standing horses.

OBJECTIVE: To evaluate the efficacy of 2 different oxygen delivery strategies-intranasal and tracheal insufflation-on the inspired fraction of oxygen (FIO2) in standing horses and to determine the time needed for arterial oxygen partial pressure (PaO2) equilibration. ANIMALS: 6 healthy adult horses. PROCEDURES: In this blinded, randomized crossover design study, horses were randomly assigned to receive oxygen via nasal cannula (group N) or transcutaneous tracheal catheter (group T). After placement of venous and arterial catheters, FIO2 was measured through a catheter placed into the distal portion of the trachea. After baseline measurements were obtained, horses received oxygen at up to 25 mL/kg/min for 1 hour via either intranasal or intratracheal catheter. The FIO2 and PaO2 were recorded at 5, 10, 15, 20, 25, 30, 45, and 60 minutes during and 5, 10, 15, 20, and 30 minutes after oxygen insufflation. Data were analyzed by use of a 2-way repeated measures ANOVA with Tukey-Kramer post hoc testing for pairwise comparisons (P < 0.05). RESULTS: During oxygen administration, FIO2 and PaO2 increased significantly when compared with baseline, resulting in significantly higher values for group T (37.7 ± 2.4%; 214.6 ± 18 mm Hg) than for group N (34.3 ± 3.9%; 184.1 ± 11 mm Hg). The equilibration time was less than 10 minutes. CLINICAL RELEVANCE: Intratracheal oxygen administration resulted in better oxygenation than nasal insufflation and should therefore be considered in standing horses that are experiencing severe respiratory compromise. The equilibration between FIO2 and PaO2 is rapid in adult horses.

Lobster attack induces sensitization in the sea hare, Aplysia californica.

Studies of the neural mechanisms of learning, especially of sensitization, have benefitted from extensive research on the model species, Aplysia californica (hereafter Aplysia). Considering this volume of literature on mechanisms, it is surprising that our understanding of the ecological context of sensitization in Aplysia is completely lacking. Indeed, the widespread use of strong electric shock to induce sensitization (an enhancement of withdrawal reflexes following noxious stimulation) is completely unnatural and leaves unanswered the question of whether this simple form of learning has any ecological relevance. We hypothesized that sublethal attack by a co-occurring predator, the spiny lobster, Panulirus interruptus, might be a natural sensitizing stimulus. We tested reflex withdrawal of the tail-mantle and head of individual Aplysia before and after attack by lobsters. Lobster attack significantly increased the amplitude of both reflexes, with a temporal onset that closely matched that observed with electric shock. This result suggests that electric shock may indeed mimic at least one naturally occurring sensitizing stimulus, suggesting, for the first time, an ecological context for this well studied form of learning.

TLR4 ligands induce IFN-alpha production by mouse conventional dendritic cells and human monocytes after IFN-beta priming.

Exacerbation of disease in systemic lupus erythematosus (SLE) is associated with bacterial infection. In conventional dendritic cells (cDCs), the TLR4 ligand bacterial LPS induces IFN-beta gene expression but does not induce IFN-alpha. We hypothesized that when cDCs are primed by cytokines, as may frequently be the case in SLE, LPS would then induce the production of IFN-alpha, a cytokine believed to be important in lupus pathogenesis. In this study we show that mouse cDCs and human monocytes produce abundant IFN-alpha following TLR4 engagement whether the cells have been pretreated either with IFN-beta or with a supernatant from DCs activated by RNA-containing immune complexes from lupus patients. This TLR4-induced IFN-alpha induction is mediated by both an initial TRIF-dependent pathway and a subsequent MyD88-dependent pathway, in contrast to TLR3-induced IFN-alpha production, which is entirely TRIF-dependent. There is also a distinct requirement for IFN regulatory factors (IRFs), with LPS-induced IFN-alpha induction being entirely IRF7- and partially IRF5-dependent, in contrast to LPS-induced IFN-beta gene induction which is known to be IRF3-dependent but largely IRF7-independent. This data demonstrates a novel pathway for IFN-alpha production by cDCs and provides one possible explanation for how bacterial infection might precipitate disease flares in SLE.

Cervical Epidural Spinal Analgesia for Acute Management of Severe Unilateral Forelimb Lameness: Case Report.

A 20-year-old Quarter Horse gelding was presented with severe right forelimb lameness (5/5 AAEP Lameness Scale) due to a tear of the superficial digital flexor muscle which was diagnosed via palpation of swelling and ultrasonography revealing major muscle fiber disruption and hematoma formation. When traditional systemic therapy (non-Steroidal anti-inflammatories) did not restore clinically acceptable comfort and the risk of supporting limb laminitis became a reasonable concern, a cervical epidural catheter was placed between the first and second cervical vertebrae in the standing, sedated patient using ultrasound guidance. The gelding was treated with epidural morphine (0.1 mg/kg every 24 h then decreased to 0.05 mg/kg every 12 h) and was pain-scored serially following treatment. Spinal analgesia was provided for 3 days. Pain scores significantly decreased following each treatment with morphine, and the gelding was successfully managed through the acutely painful period without any adverse effects associated with the C1-C2 epidural catheter placement technique, the epidural morphine, or contralateral limb laminitis. At the 2-month follow-up, the gelding was walking sound with no complications seen at the catheter insertion site. In this case, spinal analgesia using epidural morphine administered via a cervical epidural catheter was an effective and technically achievable option for pain management associated with severe forelimb muscle injury in a horse.

NPM-ALK-Induced Reprogramming of Mature TCR-Stimulated T Cells Results in Dedifferentiation and Malignant Transformation.

Fusion genes including NPM-ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM-ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM-ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)-generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM-ALK translocations. These findings describe the fundamental mechanisms of NPM-ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation. SIGNIFICANCE: This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM-ALK, and delineates dynamic transcriptional changes as a T cell transforms.See related commentary by Spasevska and Myklebust, p. 3160.