Dose-Specific Intratumoral GM-CSF Modulates Breast Tumor Oxygenation and Antitumor Immunity.

GM-CSF has been employed as an adjuvant to cancer immunotherapy with mixed results based on dosage. We previously showed that GM-CSF regulated tumor angiogenesis by stimulating soluble vascular endothelial growth factor (VEGF) receptor-1 from monocytes/macrophages in a dose-dependent manner that neutralized free VEGF, and intratumoral injections of high-dose GM-CSF ablated blood vessels and worsened hypoxia in orthotopic polyoma middle T Ag (PyMT) triple-negative breast cancer (TNBC). In this study, we assessed both immunoregulatory and oxygen-regulatory components of low-dose versus high-dose GM-CSF to compare effects on tumor oxygen, vasculature, and antitumor immunity. We performed intratumoral injections of low-dose GM-CSF or saline controls for 3 wk in FVB/N PyMT TNBC. Low-dose GM-CSF uniquely reduced tumor hypoxia and normalized tumor vasculature by increasing NG2+ pericyte coverage on CD31+ endothelial cells. Priming of "cold," anti-PD1-resistant PyMT tumors with low-dose GM-CSF (hypoxia reduced) sensitized tumors to anti-PD1, whereas high-dose GM-CSF (hypoxia exacerbated) did not. Low-dose GM-CSF reduced hypoxic and inflammatory tumor-associated macrophage (TAM) transcriptional profiles; however, no phenotypic modulation of TAMs or tumor-infiltrating lymphocytes were observed by flow cytometry. In contrast, high-dose GM-CSF priming increased infiltration of TAMs lacking the MHC class IIhi phenotype or immunostimulatory marker expression, indicating an immunosuppressive phenotype under hypoxia. However, in anti-PD1 (programmed cell death 1)-susceptible BALB/c 4T1 tumors (considered hot versus PyMT), high-dose GM-CSF increased MHC class IIhi TAMs and immunostimulatory molecules, suggesting disparate effects of high-dose GM-CSF across PyMT versus 4T1 TNBC models. Our data demonstrate a (to our knowledge) novel role for low-dose GM-CSF in reducing tumor hypoxia for synergy with anti-PD1 and highlight why dosage and setting of GM-CSF in cancer immunotherapy regimens require careful consideration.

Tetramerization of STAT5 regulates monocyte differentiation and the dextran sulfate sodium-induced colitis in mice.

In response to external stimuli during immune responses, monocytes can have multifaceted roles such as pathogen clearance and tissue repair. However, aberrant control of monocyte activation can result in chronic inflammation and subsequent tissue damage. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces monocyte differentiation into a heterogenous population of monocyte-derived dendritic cells (moDCs) and macrophages. However, the downstream molecular signals that dictate the differentiation of monocytes under pathological conditions is incompletely understood. We report here that the GM-CSF-induced STAT5 tetramerization is a critical determinate of monocyte fate and function. Monocytes require STAT5 tetramers to differentiate into moDCs. Conversely, the absence of STAT5 tetramers results in a switch to a functionally distinct monocyte-derived macrophage population. In the dextran sulfate sodium (DSS) model of colitis, STAT5 tetramer-deficient monocytes exacerbate disease severity. Mechanistically, GM-CSF signaling in STAT5 tetramer-deficient monocytes results in the overexpression of arginase I and a reduction in nitric oxide synthesis following stimulation with lipopolysaccharide. Correspondingly, the inhibition of arginase I activity and sustained supplementation of nitric oxide ameliorates the worsened colitis in STAT5 tetramer-deficient mice. This study suggests that STAT5 tetramers protect against severe intestinal inflammation through the regulation of arginine metabolism.

Tetramerization of STAT5 promotes autoimmune-mediated neuroinflammation.

Signal tranducer and activator of transcription 5 (STAT5) plays a critical role in mediating cellular responses following cytokine stimulation. STAT proteins critically signal via the formation of dimers, but additionally, STAT tetramers serve key biological roles, and we previously reported their importance in T and natural killer (NK) cell biology. However, the role of STAT5 tetramerization in autoimmune-mediated neuroinflammation has not been investigated. Using the STAT5 tetramer-deficient Stat5a-Stat5b N-domain double knockin (DKI) mouse strain, we report here that STAT5 tetramers promote the pathogenesis of experimental autoimmune encephalomyelitis (EAE). The mild EAE phenotype observed in DKI mice correlates with the impaired extravasation of pathogenic T-helper 17 (Th17) cells and interactions between Th17 cells and monocyte-derived cells (MDCs) in the meninges. We further demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated STAT5 tetramerization regulates the production of CCL17 by MDCs. Importantly, CCL17 can partially restore the pathogenicity of DKI Th17 cells, and this is dependent on the activity of the integrin VLA-4. Thus, our study reveals a GM-CSF-STAT5 tetramer-CCL17 pathway in MDCs that promotes autoimmune neuroinflammation.

Ampicillin pharmacokinetics in azotemic and healthy dogs.

BACKGROUND: Little is known about effects of factors such as kidney disease, affecting ampicillin pharmacokinetics in dogs. OBJECTIVES: Determine the pharmacokinetics of ampicillin after a single intravenous dose in healthy and azotemic dogs. ANIMALS: Nine dogs presenting with acute kidney injury and 10 healthy dogs. METHODS: This was a prospective study. An ampicillin dose of 22.2 mg/kg (mean dose) was administered once intravenously. Blood samples were obtained at timed intervals (just before administration, 1, 2, 4, 12, and 24 hours), analyzed using high-pressure liquid chromatography followed by pharmacokinetic analysis of the plasma drug concentrations. RESULTS: Peak ampicillin concentration (mcg/mL; 97.07 (36.1) vs 21.3 (50.26)), P<.001 (geometric mean (coefficient of variation, CV%)), half-life (hours; 5.86 (56.55) vs 0.97 (115.3)), P<.001) and AUC (h × mcg/mL; 731.04 (83.75) vs 33.57 (53.68)), P<.001) were greater in azotemic dogs than in healthy dogs. Azotemic dogs also had significantly lower clearance (30.06 (84.19) vs 655.03 (53.67); mL/kg h, P < .001) and volume of distribution (253.95 (30.14) vs 916.93 (135.24); mL/kg, P <.001) compared to healthy dogs. CONCLUSION AND CLINICAL IMPORTANCE: Increased drug concentrations and slower clearance of ampicillin in azotemic dogs could have clinical importance in contributing to antibiotic associated morbidity requiring indicating the need to adjust ampicillin dosing in dogs with decreased kidney function.

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment.

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived  T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

The Role of Granulocyte-Macrophage Colony-Stimulating Factor in Murine Models of Multiple Sclerosis.

Multiple sclerosis (MS) is an immune-mediated disease that predominantly impacts the central nervous system (CNS). Animal models have been used to elucidate the underpinnings of MS pathology. One of the most well-studied models of MS is experimental autoimmune encephalomyelitis (EAE). This model was utilized to demonstrate that the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical and non-redundant role in mediating EAE pathology, making it an ideal therapeutic target. In this review, we will first explore the role that GM-CSF plays in maintaining homeostasis. This is important to consider, because any therapeutics that target GM-CSF could potentially alter these regulatory processes. We will then focus on current findings related to the function of GM-CSF signaling in EAE pathology, including the cell types that produce and respond to GM-CSF and the role of GM-CSF in both acute and chronic EAE. We will then assess the role of GM-CSF in alternative models of MS and comment on how this informs the understanding of GM-CSF signaling in the various aspects of MS immunopathology. Finally, we will examine what is currently known about GM-CSF signaling in MS, and how this has promoted clinical trials that directly target GM-CSF.

Monocytes and Monocyte-Derived Antigen-Presenting Cells Have Distinct Gene Signatures in Experimental Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease mediated by a complex interaction between the autoreactive lymphocytes and the effector myeloid cells within the central nervous system (CNS). In a murine model of MS, experimental autoimmune encephalomyelitis (EAE), Ly6Chi monocytes migrate into the CNS and further differentiate into antigen-presenting cells (APCs) during disease progression. Currently, there is no information about gene signatures that can distinguish between monocytes and the monocyte-derived APCs. We developed a surface marker-based strategy to distinguish between these two cell types during the stage of EAE when the clinical symptoms were most severe, and performed transcriptome analysis to compare their gene expression. We report here that the inflammatory CNS environment substantially alters gene expression of monocytes, compared to the monocyte differentiation process within CNS. Monocytes in the CNS express genes that encode proinflammatory cytokines and chemokines, and their expression is mostly maintained when the cells differentiate. Moreover, monocyte-derived APCs express surface markers associated with both dendritic cells and macrophages, and have a significant up-regulation of genes that are critical for antigen presentation. Furthermore, we found that Ccl17, Ccl22, and Ccr7 are expressed in monocyte-derived APCs but not the Ly6Chi monocytes. These findings may shed light on identifying molecular signals that control monocyte differentiation and functions during EAE.

Ischemic stroke alters immune cell niche and chemokine profile in mice independent of spontaneous bacterial infection.

INTRODUCTION: Stroke-associated pneumonia (SAP) is a major cause of mortality in patients who have suffered from severe ischemic stroke. Although multifactorial in nature, stroke-induced immunosuppression plays a key role in the development of SAP. Previous studies using a murine model of transient middle cerebral artery occlusion (tMCAO) have shown that focal ischemic stroke induction results in functional defects of lymphocytes in the spleen, thymus, and peripheral blood, leading to spontaneous bacterial infection in the lungs without inoculation. However, how ischemic stroke alters immune cell niche and the expression of cytokines and chemokines in the lungs has not been fully characterized. METHODS: Ischemic stroke was induced in mice by tMCAO. Immune cell profiles in the brain and the lungs at 24- and 72-hour time points were compared by flow cytometric analysis. Cytokine and chemokine expression in the lungs were determined by multiplex bead arrays. Tissue damage and bacterial burden in the lungs following tMCAO were evaluated. RESULTS: Ischemic stroke increases the percentage of alveolar macrophages, neutrophils, and CD11b+ dendritic cells, but reduces the percentage of CD4+ T cells, CD8+ T cells, B cells, natural killer cells, and eosinophils in the lungs. The alteration of immune cell niche in the lungs coincides with a significant reduction in the levels of multiple chemokines in the lungs, including CCL3, CCL4, CCL5, CCL17, CCL20, CCL22, CXCL5, CXCL9, and CXCL10. Spontaneous bacterial infection and tissue damage following tMCAO, however, were not observed. CONCLUSION: This is the first report to demonstrate a significant reduction of lymphocytes and multiple proinflammatory chemokines in the lungs following ischemic stroke in mice. These findings suggest that ischemic stroke directly impacts pulmonary immunity.

Effect of urinary bladder lavage on in-hospital recurrence of urethral obstruction and durations of urinary catheter retention and hospitalization for male cats.

OBJECTIVE To evaluate the effect of urinary bladder lavage on in-hospital recurrence of urethral obstruction (UO) and durations of urinary catheter retention and hospitalization for male cats. DESIGN Randomized controlled clinical trial. ANIMALS 137 male cats with UO. PROCEDURES Following random allocation, cats either did (flush group; n = 69) or did not (no-flush group; 68) undergo urinary bladder lavage with saline (0.9% NaCl) solution after alleviation of the obstruction and placement of a urethral catheter. Signalment, prior history of UO, presence of crystalluria, difficulty of urinary tract catheterization, in-hospital UO recurrence rate, and durations of urinary catheter retention and hospitalization were compared between the flush and no-flush groups. RESULTS Baseline characteristics did not differ significantly between the 2 treatment groups. The in-hospital UO recurrence rate (9/69 [13%]) and median durations of urinary catheter retention (37 hours; range, 3 to 172 hours) and hospitalization (3 days; range, 0.5 to 12 days) for the flush group did not differ significantly from the in-hospital UO recurrence rate (13/68 [19%]) and median durations of urinary catheter retention (36 hours; range, 1 to 117 hours) and hospitalization (3 days; range, 1 to 9 days) for the no-flush group. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that, for male cats with UO, urinary bladder lavage at the time of urethral catheterization had no significant effect on in-hospital recurrence rate of the condition, duration of urinary catheter retention, or duration of hospitalization; however, additional studies are necessary to validate or refute these findings.

Cesarean delivery rates among family physicians versus obstetricians: a population-based cohort study using instrumental variable methods.

BACKGROUND: Previous research suggests that family physicians have rates of cesarean delivery that are lower than or equivalent to those for obstetricians, but adjustments for risk differences in these analyses may have been inadequate. We used an econometric method to adjust for observed and unobserved factors affecting the risk of cesarean delivery among women attended by family physicians versus obstetricians. METHODS: This retrospective population-based cohort study included all Canadian (except Quebec) hospital deliveries by family physicians and obstetricians between Apr. 1, 2006, and Mar. 31, 2009. We excluded women with multiple gestations, and newborns with a birth weight less than 500 g or gestational age less than 20 weeks. We estimated the relative risk of cesarean delivery using instrumental-variable-adjusted and logistic regression. RESULTS: The final cohort included 776 299 women who gave birth in 390 hospitals. The risk of cesarean delivery was 27.3%, and the mean proportion of deliveries by family physicians was 26.9% (standard deviation 23.8%). The relative risk of cesarean delivery for family physicians versus obstetricians was 0.48 (95% confidence interval [CI] 0.41-0.56) with logistic regression and 1.27 (95% CI 1.02-1.57) with instrumental-variable-adjusted regression. INTERPRETATION: Our conventional analyses suggest that family physicians have a lower rate of cesarean delivery than obstetricians, but instrumental variable analyses suggest the opposite. Because instrumental variable methods adjust for unmeasured factors and traditional methods do not, the large discrepancy between these estimates of risk suggests that clinical and/or sociocultural factors affecting the decision to perform cesarean delivery may not be accounted for in our database.

Cytokine Signaling in Multiple Sclerosis and Its Therapeutic Applications.

Multiple sclerosis (MS) is one of the most common neurological disorders in young adults. The etiology of MS is not known but it is widely accepted that it is autoimmune in nature. Disease onset is believed to be initiated by the activation of CD4+ T cells that target autoantigens of the central nervous system (CNS) and their infiltration into the CNS, followed by the expansion of local and infiltrated peripheral effector myeloid cells that create an inflammatory milieu within the CNS, which ultimately lead to tissue damage and demyelination. Clinical studies have shown that progression of MS correlates with the abnormal expression of certain cytokines. The use of experimental autoimmune encephalomyelitis (EAE) model further delineates the role of these cytokines in neuroinflammation and the therapeutic potential of manipulating their biological activity in vivo. In this review, we will first present an overview on cytokines that may contribute to the pathogenesis of MS or EAE, and provide successful examples and roadblock of translating data obtained from EAE to MS. We will then focus in depth on recent findings that demonstrate the pathological role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in MS and EAE, and briefly discuss the potential of targeting effector myeloid cells as a treatment strategy for MS.

Clinical findings, diagnostics and outcome in 33 cats with adrenal neoplasia (2002-2013).

OBJECTIVES: The objective of this retrospective study was to describe the clinical signs and diagnostic findings in cats with histopathologically confirmed adrenal neoplasms, and to assess correlations with survival data. METHODS: Study data were acquired by reviewing medical records for all cats diagnosed with adrenal neoplasms at seven referral institutions between 2002 and 2013. Inclusion criteria required a histopathologic diagnosis of an adrenal neoplasm (ante-mortem or on necropsy). RESULTS: Thirty-three cats met the inclusion criteria for the study. The most common presenting complaints included weakness (n = 12), respiratory signs (n = 4), blindness (n = 4) or gastrointestinal signs (n = 3). Laboratory abnormalities included hypokalemia (n = 18), alkalemia (n = 12), elevated creatine kinase (>3000, n = 5) and azotemia (n = 4). In addition, hypertension was noted in 13 cats. Thirty cats were diagnosed with cortical tumors (17 carcinomas, 13 adenomas) and three cats were diagnosed with pheochromocytomas. Twenty-five cats underwent tests to evaluate the function of the adrenal tumors; 19/25 cats had functional tumors (hyperaldosteronism [n = 16], hypercortisolemia [n = 1], high estradiol [n = 1], and hypersecretion of aldosterone, estradiol and progesterone [n = 1]). Twenty-six cats underwent adrenalectomy, one cat was medically managed and six were euthanized without treatment. Long-term survival postoperatively ranged from 4-540 weeks, with 20 (77%) cats surviving the perioperative period of 2 weeks. The only variable that was found to be negatively associated with survival was female sex. The most common complications noted during the perioperative period were hemorrhage and progressive lethargy and anorexia. CONCLUSIONS AND RELEVANCE: Surgical treatment for feline adrenal tumors (regardless of tumor type) resulted in good long-term survival. Given that pre- and postoperative hypocortisolemia was identified in this study, and, in addition, hypersecretion of more than one adrenal hormone occurred in one cat, adrenal panels prior to surgery may be beneficial as part of the preoperative work-up.

Transient cold agglutinins associated with Mycoplasma cynos pneumonia in a dog.

This report details a case of reversible cold agglutinins in a dog with Mycoplasma cynos pneumonia. An 11-month-old female spayed Rhodesian Ridgeback was presented for lethargy and cough. Thoracic radiographs revealed an alveolar pattern present bilaterally in the cranioventral lung lobes. Septic neutrophilic inflammation with suspected Mycoplasma sp. organisms was noted on cytologic examination of a trans-tracheal wash, and the dog was treated empirically with IV ampicillin/sulbactam and enrofloxacin pending culture results. Red blood cell agglutination was noted unexpectedly on several blood film reviews during hospitalization; however, the dog never developed clinical or laboratory evidence of hemolysis. Cold agglutinins were demonstrated based on the results of a saline dilution and cold agglutinin test that showed agglutination at 4°C but not at room temperature (21°C) or 37°C. Based on a positive culture for M cynos, the dog was treated for 8 weeks with oral enrofloxacin. After clinical and radiographic resolution of the pneumonia, repeated saline dilution and cold agglutinin tests of peripheral blood were negative at all temperatures. Reversible, asymptomatic cold agglutinins are common in human patients with mycoplasma pneumonia, but this is the first reported case in a dog.

Feline acute kidney injury: 1. Pathophysiology, etiology and etiology-specific management considerations.

PRACTICAL RELEVANCE: Acute kidney injury (AKI) is a frequently recognized disease process in cats that requires immediate and aggressive intervention. A thorough understanding of the pathophysiologic processes underlying AKI and familiarity with the most common etiologies are essential for providing the most effective and timely therapy. Possessing this knowledge will also allow a more accurate prognosis to be given, and afford the best chance of a favorable outcome. CLINICAL CHALLENGES: Feline patients often present with vague signs of AKI, which may delay treatment and adversely affect the prognosis. Their response to injury and treatment is often different to that of other species. AUDIENCE: This two-part review article is directed at small animal practitioners as well as specialists. Part 1 reviews mechanisms underlying AKI in the cat, as well as etiologies and treatments related to some specific causes of AKI. EVIDENCE BASE: The veterinary literature is limited with regards to the pathophysiology of AKI unique to the cat. However, there are numerous feline studies evaluating causes of AKI.

Feline acute kidney injury: 2. Approach to diagnosis, treatment and prognosis.

PRACTICAL RELEVANCE: Feline acute kidney injury (AKI) is a commonly recognized problem in small animal practice that requires prompt diagnosis and directed therapy. There are many treatment methods with which practitioners should be familiar, including medical options, surgical interventions and renal replacement therapy (dialysis). It is important to know which option is most appropriate for each cause and stage of AKI to deliver the most effective therapy. CLINICAL CHALLENGES: AKI can cause vague clinical signs, but a vast array of life-threatening sequelae. Rapid recognition of potential complications and knowledge of treatment options is imperative for successful management. Feline patients also require an understanding of their unique physiology as it relates to the therapeutic plan. AUDIENCE: This two-part review article is directed at small animal practitioners as well as specialists. Part 2 discusses the diagnosis of AKI in cats using physical examination findings, clinicopathologic results and imaging modalities. The treatment of AKI and its sequelae is also reviewed, with information on recent advances in this area. EVIDENCE BASE: While there is very limited data comparing the outcomes of various treatment options, there is literature addressing the use of several medications, as well as renal replacement therapy, in cats.

Extracorporeal removal of drugs and toxins.

Renal replacement therapies (RRT) are increasingly used for the treatment of acute and chronic kidney diseases as well as intoxications and accidental drug overdoses. These therapies offer a mechanism for the removal of toxic substances from the patient's blood and supplement the standard detoxification protocols. If instituted early, RRT can have a significant effect on the course of the toxicity; however, this process is not selective for the removal of only harmful products and can also result in the clearance of medications intended for therapeutic use.