Marco Franchini

Cytokine induction in pulmonary airways of horses with heaves and effect of therapy with inhaled fluticasone propionate

Work in humans and laboratory animals has identified a central role for cytokines and chemokines in development and persistence of lower airway inflammation. The objectives of this study were to determine interleukin (IL)-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha induction in bronchoalveolar lavage (BAL) of control horses and horses with heaves both during remission and exacerbation of the disease, and to determine the effect of therapy with inhaled fluticasone propionate on the cytokine profile of horses with heaves. IL-1 beta and TNF-alpha mRNA expression was significantly higher in horses with heaves after exposure to moldy hay compared to either values obtained during clinical remission or to healthy controls. IL-8 mRNA expression and protein concentrations were significantly higher in horses with heaves than in controls. Both IL-4 and IFN-gamma mRNA expression was increased at various times in heaves-susceptible horses compared to controls. IL-2, IL-5 and IL-10 mRNA expression was not detected in BAL cells of either group. Therapy with inhaled fluticasone propionate after induction of a severe heaves exacerbation resulted in complete resolution of clinical signs, normalization of pulmonary function tests, and significant decrease in BAL neutrophilia. This was associated with a significant decrease in IL-4 mRNA expression and increase in IFN-gamma/IL-4 ratio in horses with heaves. These results demonstrate the clinical efficacy of inhaled fluticasone propionate for the treatment of heaves and suggest a role for cytokines in the development of lower airway inflammation in heaves-susceptible horses.

The role of neutrophil chemotactic cytokines in the pathogenesis of equine chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is defined as a chronic obstructive inflammatory disease affecting the small airways associated with hay dust exposure (Lowell, F.C., 1964. Observation on heaves. An asthma like syndrome in the horse, J. Allergy 35, 322-330). The disease corresponds histopathologically to a chronic bronchiolitis (Gerber, H., 1973. Chronic pulmonary disease in the horse, Equine Vet. J. 5, 26-33; Winder, N.C., Grünig, G., Hermann, M., Howald, B., von Fellenberg, R., 1989. Comparison of respiratory secretion cytology and pulmonary histology in horses, J. Vet. Med., A36, 32-38) and is mainly characterized by the presence of neutrophil granulocytes in the small bronchioles. Around 12-50% of all horses in Europe and the northern United States suffer from this disease (Mc Pherson, E.A., Lawson, G.H.K., Murphy, J.R., Nicholson, J.M., Fraser, J.A., Breeze, R.G., Pirie, H.M., 1978. Chronic obstructive pulmonary disease (COPD): Identification of affected horses, Eq. Vet. J. 10, 47-53; Larson, V.L., Busch, R.H., 1985. Equine tracheobronchial lavage: Comparison of lavage cytologic features in horses with chronic obstructive pulmonary disease, Am. J. Vet. Res., 46, 144-146; Bracher, V., von Fellenberg, R., Winder, N.C., Grünig, G., 1991. An investigation of the incidence of chronic obstructive pulmonary disease (COPD) in random populations of swiss horses, Equine Vet. J. 23, 136-141). The number of neutrophils in the bronchoalveolar lavage (BAL) and in tracheobronchial secretions (TBS) correlates with the severity of the disease. The present study is focused on the mechanisms which lead to the infiltration of neutrophil granulocytes in the lung of horses. We found that: (1). A strong chemotactic activity in the BAL fluid is associated with high levels of dust exposition. (2). In vitro stimulated alveolar macrophages have impaired phagocytosis efficiency and secrete two chemo-attractants specific for neutrophil granulocytes: Interleukin-8 (IL-8) (Wuyts, A., Proost, P., Put, W., Lenaerts, J.-P., Paemen, L., van Damme, J., 1994. Leucocyte recruitment by monocyte chemotactic proteins (MCPs) secreted by human phagocytes, J. Immunol. Meth. 174, 237-247) and macrophage inflammatory protein-2 (MIP-2) (Wolpe, S.D., Sherry, B., Juers, D., Davatelis, G. Yurt, R.W., Cerami, A., Identification and characterisation of macrophage inflammatory protein-2, Proc. Natl. Acad. Sci. USA 86, 612-616; Tekamp-Olson, P., Gallegos, C., Bauer, D., 1990. Cloning and characterisation of cDNAs for murine macrophage inflammatory protein-2 and its human homologues, J. Exp. Med., 172, 911-927; Driscoll, K.E., 1994. Macrophage inflammatory proteins: Biology and role in pulmonary inflammation. Exp. Lung Res., 20, 473-490). This is associated with the appearance of chemotactic activity in the supernatant. These data confirmed our working hypothesis that bronchiolar neutrophilia may be the consequence of a (over)stimulation of pulmonary macrophages leading to expression of cytokines chemotactic for neutrophil granulocytes.

Interplay between Alpha/Beta and Gamma Interferons with B, T, and Natural Killer Cells in the Defense against Herpes Simplex Virus Type 1

ABSTRACT The essential components of the immune system that control primary and chronic infection with herpes simplex virus type 1 (HSV-1) in mice were investigated. Infection within the first few days can be controlled by alpha/beta interferon (IFN-α/β) alone without significant contribution of B, T, or NK cells. IFN-α/β and IFN-γ cooperate in the elimination of virus in the absence of these lymphocytes. In contrast, B, T, or NK cells appear to be required to control persistent infection with HSV-1. These results suggest that distinct and essential immune elements are recruited in a time-dependent fashion to control acute and persistent HSV-1 infection.

Flt3 Ligand–treated Neonatal Mice Have Increased Innate Immunity Against Intracellular Pathogens and Efficiently Control Virus Infections

Flt-3 ligand (FL), a hematopoetic growth factor, increases the number of dendritic cells (DCs), B cells, and natural killer cells in adult mice but the effect in neonates was unknown. We show that FL treatment of newborn mice induced a >100-fold increase in the innate resistance against infection with herpes simplex virus type 1 and Listeria monocytogenes. This resistance required interferon (IFN)-α/β for viral and interleukin (IL)-12 for bacterial infections. Long-term survival after viral but not bacterial infection was increased ∼100-fold by FL treatment. After treatment, CD11c+/major histocompatibility complex type II+ and CD11c+/B220+ DC lineage cells were the only cell populations increased in the spleen, liver, peritoneum, and skin. DC induction was independent of IFNs, IL-2, -4, -7, -9, -15, and mature T and B cells. The data suggest that FL increases the number of DCs in neonates and possibly in other immune-compromised individuals, which in turn improves IFN-α/β– and IL-12–associated immune responses.

Both Viral and Host Factors Contribute to Neurovirulence of Bovine Herpesviruses 1 and 5 in Interferon Receptor-Deficient Mice

ABSTRACT Herpes simplex virus (HSV) type 1 and bovine herpesviruses 1 and 5 (BHV-1 and BHV-5) can use the same cellular receptor for entry, but only HSV is known to cause disease in mice. We hypothesized that components of either the innate or the adaptive immune system, or a combination of both, were responsible for curbing replication of BHVs in mice. Therefore, wild-type mice as well as mice with various combined genetic deficiencies in the alpha/beta interferon receptor or gamma interferon receptor and in the ability to produce mature B and T lymphocytes (RAG-2 deletion) were infected with BHV-1 and BHV-5 and monitored clinically, serologically, histopathologically, and virologically. A functional immune system protected the mice from disease and death due to BHV infection, and the immune response was Th1 like. BHV-5 was transported to the central nervous system by the axonal pathway, whereas viremia was required for this outcome with BHV-1. The alpha/beta interferon system was able to obstruct quantitative spread of the viruses in the infected organism. The gamma interferon system had a protective effect against BHV-1, even in mice with the RAG-2 deletion. In contrast, the same mice succumbed to neurological disease and death upon infection with BHV-5. Productively infected neurons were detected only in BHV-5-infected mice with an intact gamma interferon system. We conclude that the alpha/beta interferon system had a protective effect, while an intact gamma interferon system was required for efficient replication of BHV-5 in mouse neurons and for the development of neurological disease.

Predictors of clinical remission in cats with diabetes mellitus.

BACKGROUND Clinical remission is frequent in cats with well-controlled diabetes mellitus, but few studies explored predictors of this phenomenon. HYPOTHESIS Data retrieved from medical records at admission might be valuable to identify likelihood of remission and its duration in diabetic cats. ANIMALS Ninety cats with newly diagnosed diabetes, followed-up until death or remission. METHODS Retrospective cohort study. Data were collected from records at admission, including history, signalment, physical examination, haematology, and biochemical profile, and the occurrence and duration of remission, defined as normoglycemia without insulin for ≥4 weeks. Predictors of remission were studied with univariate and multivariate logistic regression. Factors associated with remission duration were analyzed with Kaplan-Meier and Cox proportional hazard models. RESULTS Forty-five (50%) cats achieved remission, after a median time of 48 days (range: 8-216). By study end, median remission duration was 114 days (range: 30-3,370) in cats that died and 151 days (range: 28-1,180) in alive cats. Remission was more likely with higher age (OR: 1.23, 95% CI: 1.04-1.46; P=.01) and less likely with increased serum cholesterol (OR: 0.36, 95% CI: 0.11-0.87; P=.04). Remission was longer with higher body weight (HR: 0.65, 95% CI: 0.42-0.99; P=.04) and shorter with higher blood glucose (HR: 1.01, 95% CI: 1.00-1.02; P=.02). CONCLUSIONS AND CLINICAL IMPORTANCE Age, body weight, cholesterol, and glucose levels are suggested for prediction of remission or its duration in diabetic cats. Older cats developing diabetes may have a better outcome, possibly suggesting a slower disease progression.

Interleukin-12- and Gamma Interferon-Dependent Innate Immunity Are Essential and Sufficient for Long-Term Survival of Passively Immunized Mice Infected with Herpes Simplex Virus Type 1

ABSTRACT Interferon (IFN) type I (alpha/beta IFN [IFN-α/β]) is very important in directly controlling herpes simplex virus type I (HSV-1) replication as well as in guiding and upregulating specific immunity against this virus. By contrast, the roles of IFN type II (IFN-γ) and antibodies in the defense against HSV-1 are not clear. Mice without a functional IFN system and no mature B and T cells (AGR mice) did not survive HSV-1 infection in the presence or absence of neutralizing antibodies to the virus. Mice without a functional IFN type I system and with no mature B and T cells (AR129 mice) were unable to control infection with as little as 10 PFU of HSV-1 strain F. By contrast, in the presence of passively administered neutralizing murine antibodies to HSV-1, some AR129 mice survived infection with up to104PFU of HSV-1. This acute immune response was dependent on the presence of interleukin-12 (IL-12) p75. Interestingly, some virus-infected mice stayed healthy for several months, at which time antibody to HSV-1 was no longer detectable. Treatment of these virus-exposed mice with dexamethasone led to death in approximately 40% of the mice. HSV-1 was found in brains of mice that did not survive dexamethasone treatment, whereas HSV-1 was absent in those that survived the treatment. We conclude that in the presence of passively administered HSV-1-specific antibodies, the IL-12-induced IFN-γ-dependent innate immune response is able to control low doses of virus infection. Surprisingly, in a significant proportion of these mice, HSV-1 appears to persist in the absence of antibodies and specific immunity.

Evaluation of a novel real-time continuous glucose-monitoring system for use in cats

BACKGROUND The Guardian REAL-Time is a continuous glucose-monitoring system (CGMS) recently developed to provide instantaneous interstitial glucose concentrations; the system does not require a monitor being fixed to the animal. HYPOTHESIS The CGMS provides accurate and reproducible real-time readings of glucose concentration in cats. ANIMALS Thirty-two diabetic cats, 2 cats with suspected insulinoma, and 5 healthy cats. METHODS Prospective, observational study. CGMS accuracy was compared with a reference glucose meter at normal, high, and low blood glucose concentrations using error grid analysis. Reading variability of 2 simultaneously used CGMS was determined in diabetic cats by calculating correlation and percentage of concordance of paired data at different glycemic ranges. The time interval between increasing glycemia and a rise in interstitial fluid glucose measured by the CGMS was assessed in healthy cats receiving glucose IV; the time point of maximal increase in interstitial glucose concentrations was calculated. RESULTS The CGMS was 100, 96.1, and 91.0% accurate at normal, high, and low blood glucose concentrations. Measurements deviated from reference by -12.7 +/- 70.5 mg/dL at normal, -12.1 +/- 141.5 mg/dL at high, and -1.9 +/- 40.9 mg/dL at low glucose concentrations. Overall, paired CGMS readings correlated significantly (r = 0.95, P < .0001) and concordance was 95.7%. The median delay after IV administration of glucose to an increase in interstitial glucose was 11.4 minutes (range: 8.8-19.7 minutes). CONCLUSIONS AND CLINICAL IMPORTANCE Although some readings substantially deviated from reference values, the CGMS yields reproducible results, is clinically accurate in cats with hyperglycemia and euglycemia, and is slightly less accurate if blood glucose concentrations are low. Rapidly increasing interstitial glucose after a glycemic rise suggests that the CGMS is suitable for real-time measurement under clinical conditions.

Dendritic Cells from Mice Neonatally Vaccinated with Modified Vaccinia Virus Ankara Transfer Resistance against Herpes Simplex Virus Type I to Naive One-Week-Old Mice

Modified vaccinia Ankara (MVA) is an attenuated virus. MVA induces the production of IFN and Flt3-L (FL), which results in the expansion of dendritic cells (DC) and enhanced resistance against viral infections. We report on the interplay among IFN, FL, and DC in the resistance against heterologous virus after injection of neonatal mice with MVA. The induction of serum FL was tested on day 2, and the expansion of DC was tested 1 wk after treatment with MVA. At this time point the resistance against infection with heterologous virus was also determined. After MVA treatment, serum FL was enhanced, and DC, including plasmacytoid cells in spleen, were increased in number. Mice that lacked functional IFN type I and II systems failed to increase both the concentration of FL and the number of DC. Treatment with MVA enhanced resistance against HSV-1 in wild-type animals 100-fold, but animals without a functional IFN system were not protected. Transfer of CD11c+ cells from MVA-treated mice into naive animals protected against lethal infection with HSV-1. Thus, although the increased resistance could be largely attributed to the increase in activation of IFN-producing plasmacytoid cells, this, in turn, depends on a complex interplay between the DC and T cell systems involving both FL and IFNs.

Subacute Endotoxemia Induces Adipose Inflammation and Changes in Lipid and Lipoprotein Metabolism in Cats

Acute inflammation in humans is associated with transient insulin resistance (IR) and dyslipidemia. Chronic low-grade inflammation is a pathogenic component of IR and adipose tissue dysfunction in obesity-induced type 2 diabetes. Because feline diabetes closely resembles human type 2 diabetes, we studied whether lipopolysaccharide (LPS)-induced subacute inflammation, in the absence of obesity, is the potential primary cause of IR and metabolic disorders. Cats received increasing iv doses (10-1000 ng/kg(-1) · h(-1)) of LPS (n = 5) or saline (n = 5) for 10 d. Body temperature, proinflammatory and metabolic markers, and insulin sensitivity were measured daily. Tissue mRNA and protein expression were quantified on d 10. LPS infusion increased circulating and tissue markers of inflammation. Based on the homeostasis model assessment, endotoxemia induced transient IR and β-cell dysfunction. At the whole-body level, IR reverted after the 10-d treatment; however, tissue-specific indications of IR were observed, such as down-regulation of adipose glucose transporter 4, hepatic peroxisome proliferative activated receptor-γ1 and -2, and muscle insulin receptor substrate-1. In adipose tissue, increased hormone-sensitive lipase activity led to reduced adipocyte size, concomitant with increased plasma and hepatic triglyceride content and decreased total and high-density lipoprotein cholesterol levels. Prolonged LPS-induced inflammation caused acute IR, followed by long-lasting tissue-specific dysfunctions of lipid-, glucose-, and insulin metabolism-related targets; this ultimately resulted in dyslipidemia but not whole-body IR. Endotoxemia in cats may provide a promising model to study the cross talk between metabolic and inflammatory responses in the development of adipose tissue dysfunction and IR.