Molecular detection of Chlamydia abortus in a stranded Mediterranean striped dolphin Stenella coeruleoalba.

This study reports gross, histopathological, and molecular features of a Chlamydia abortus infection in a stranded female striped dolphin Stenella coeruleoalba from the Tyrrhenian coast of southern Italy. Post-mortem examination revealed liver congestion, splenic lymphoid depletion with capsular petechiae, and pneumonia. Histology revealed disseminated intravascular coagulation with vasculitis and congestion. Hepatocellular and acute myocardial degeneration were also observed. Basophilic, coccobacillary inclusions consistent with Chlamydia spp. were observed histologically in the type II pneumocytes, myocardial fibers, and hepatocytes, and in macrophages and plasma cells of liver, spleen, and prescapular lymph nodes. Chlamydial antigen was detected by immunofluorescence assay using genus-specific anti-Chlamydia antibodies. PCR assay revealed C. abortus in spleen, liver, heart, and lungs. C. abortus was the only pathogen detected. The main pathological changes suggest that Chlamydia infection may have been the cause of stranding and death of the striped dolphin. This case represents the first molecular detection of a member of the Chlamydiaceae in a marine mammal.

Tamoxifen induces apoptotic neutrophil efferocytosis in horses.

Macrophages and neutrophils are important cellular components in the process of acute inflammation and its subsequent resolution, and evidence increasingly suggests that they play important functions during the resolution of chronic, adaptive inflammatory processes. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including allergic asthma and chronic obstructive pulmonary disease in humans, and equine asthma (also known as recurrent airway obstruction (RAO). Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction of BALF neutrophils, and improvement in animals' clinical status. The aim of this study was to describe if TX induces in vitro efferocytosis of neutrophils by alveolar macrophages. Efferocytosis assay, myeloperoxidase (MPO) detection and translocation phosphatidylserine (PS) were performed on neutrophils isolated from peripheral blood samples from five healthy horses. In in vitro samples from heathy horses, TX treatment increases the phenomenon of efferocytosis of peripheral neutrophils by alveolar macrophages. Similar increases in supernatant MPO concentration and PS translocation were observed in TX-treated neutrophils, compared to control cells. In conclusion, these results confirm that tamoxifen has a direct effect on equine peripheral blood neutrophils, through stimulation of the engulfment of apoptotic neutrophils by alveolar macrophages.

Aspergillus fumigatus-sensitive IgE is associated with bronchial hypersensitivity in a murine model of neutrophilic airway inflammation.

Neutrophils are the predominant inflammatory cells that infiltrate airways during acute exacerbation of asthma. The importance of A. fumigatus sensitization, and IgE response in the airways in patients with acute asthma is unclear. Rockefeller (RK) mice were sensitized with A. fumigatus extract protein. The animals were subsequently challenged with different degrees of A. fumigatus contamination in the cage bedding. All groups of mice were euthanized to obtain bronchoalveolar lavage fluid (BALF) for cytological and Elisa assays, and lung tissue for histological analysis. Moreover, several bioassays were conducted to determine whether BALF IgE antibodies can activate mast cells. In this study, we demonstrated that exposure of sensitized mice to a known concentration of A. fumigatus conidia produces bronchial hyperreactivity with marked neutrophilic bronchial infiltration and increased BALF IgE, capable of triggering mast cell degranulation. This study suggests that IgE may play a role in bronchial hyperreactivity associated to A. fumigatus exposure in mice. Mice sensitized and challenged with this fungus showed characteristics of severe asthma, with an increase of BALF neutrophils, histological changes consistent with severe asthma and an increase of IgE capable of triggering type I hypersensitivity.

Pathology and molecular analysis of Hapalotrema mistroides (Digenea: Spirorchiidae) infecting a Mediterranean loggerhead turtle Caretta caretta.

Turtle blood flukes belonging to the family Spirorchiidae (Digenea) represent a major threat for sea turtle health and are considered the most important parasitic cause of turtle stranding and mortality worldwide. Despite the large diversity of spirorchiid species found globally, there are only 2 records for free-ranging Mediterranean sea turtles that date back to the late 1800s involving just Hapalotrema mistroides Monticelli, 1896. This study describes the first fatal confirmed case of spirorchiidiasis in a free-ranging Mediterranean loggerhead turtle Caretta caretta (Linnaeus) and, owing to the complexities of taxonomic identification of these parasites, provides the first molecular characterization and phylogenetic analysis of H. mistroides from the Mediterranean Sea. The loggerhead turtle showed cachexia and digestive disorders associated with severe damage to the pancreas and intestinal ganglia, caused by deposition of Hapalotrema eggs forming granulomas. Massive Hapalotrema egg emboli in several tissues and organs and encephalitis were the most probable contributions to the death of the turtle. The congruence between the phylogenetic analysis of both the ITS2 and 28S rDNA resolved the Italian and USA H. mistroides as the same species, confirming the parasite identification. The case here described clearly indicates that the blood flukes should be considered in the differential diagnosis of Mediterranean sea turtle diseases.

In Vitro effects of tamoxifen on equine neutrophils.

Neutrophils participate in innate immunity as the first line of host defense against microorganisms. However, exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including allergic asthma and chronic obstructive pulmonary disease in humans, and equine asthma (also known as recurrent airway obstruction (RAO). Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous preliminary studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction of BALF neutrophils, and improvement in animals' clinical status. The aim of this study was to evaluate the in vitro effect of TX on functional tests in equine peripheral blood neutrophils. Chemotaxis, respiratory burst production and phagocytosis assays were performed on neutrophils isolated from peripheral blood samples from 10 healthy horses. Results showed that IL-8 stimulated cells decrease their chemotactic index when treated with TX (1 and 10µM). Respiratory burst production was also dampened after treatment with TX. In conclusion, these results confirm that tamoxifen has a direct action on equine peripheral blood neutrophils. However, more in vivo and in vitro studies are required to fully understand the mechanisms of action of TX on neutrophils, in order to elucidate if it can be used as treatment in disorders such as allergic asthma in humans and horses.

The occurrence and pathogenicity of Serratospiculum tendo (Nematoda: Diplotriaenoidea) in birds of prey from southern Italy.

The air sacs of free-ranging birds of prey (n= 652) from southern Italy, including 11 species of Accipitriformes and six of Falconiforms, were examined for infections with Serratospiculum tendo (Nematoda: Diplotriaenoidea). Of the 17 species of birds examined, 25 of 31 (80.6%) peregrine falcons (Falco peregrinus) from Calabria Region and a single northern goshawk (Accipiter gentilis) from Campania Region were infected with S. tendo, suggesting a strong host specificity for the peregrine falcon. The northern goshawk and 18 of 25 infected peregrine falcons showed cachexia and all infected birds had bone fractures. At gross examination, air sacculitis and pneumonia were the most common lesions in infected birds. Microscopically, the air-sac walls showed thickening of the smooth muscle cells, resulting in a papillary appearance, along with hyperplasia of the mesothelium and epithelium, and foci of plasma cell infiltration and macrophages associated with several embryonated eggs and adult parasites. Extensive areas of inflammation were found in the lungs, characterized by lymphocytes, macrophages and fibroblasts surrounding embryonated eggs. The northern goshawk also had detachment of the dextral lung with several necrotic foci. In this case, the death of the bird was directly attributed to S. tendo infection. Lesions and pathological changes observed here suggest that S. tendo can cause disease.

Inhibition of ß1 integrin and IL-3Rß common subunit interaction hinders tumour angiogenesis.

Integrin/cytokine receptor interaction provides permissive signals leading to neoangiogenesis, and integrins are crucial for differentiation of endothelial progenitor cells (EPCs). It is known that the inflammatory interleukin-3 (IL-3), released in the tumoral microenvironment, contributes to both angiogenesis and vasculogenic processes. Herein, we generated IL-3 receptor beta common (IL-3Rßc) extracellular domain-derived fusion proteins (Fc) to elucidate the molecular mechanisms regulating these processes. Three different Fc were generated, containing the entire extracellular domain of IL-3Rßc (Fc1.4), a fragment corresponding to domains 1-3 (Fc1.3) and a fragment corresponding to domain 4 (Fc4), respectively. The ability of the fusion proteins to interfere with IL-3Rßc/ß1 integrin interaction was assessed on endothelial cells (ECs), EPCs and murine-derived ECs. Pull-down experiments showed that Fc1.4 and Fc4 fusion proteins specifically interacted with ß1 integrin. Fc4 and Fc1.4 fragments prevented IL-3-mediated EPC expansion, arterial morphogenesis and tumour-derived EC migration, without affecting cell adhesion. Fc4 in vivo inhibited the IL-3-mediated vasculogenic process, as well as inflammatory and tumour vascular growth. In conclusion, these data identify the ß1 integrin-interacting domain in the juxta-membrane IL-3Rßc extracellular domain, and provide the rational for targeting this interaction to impair vascular growth.

Does C-reactive protein identify a subclinical metabolic disease in healthy subjects?

BACKGROUND: Highly sensitive C-reactive protein (hs-CRP) levels are significant predictors of subsequent diabetes and metabolic syndrome (MS). Owing the strong correlations between components of the MS and obesity with hs-CRP levels, previous studies about the associations of hs-CRP with insulin resistance might have been confounded by the inclusion of overweight or dysmetabolic subjects. DESIGN: Our aim was to evaluate the associations between hs-CRP levels and fasting insulin and insulin resistance (evaluated by the Homeostasis Model Assessment: HOMA IR) in a subgroup of subjects with normal body mass index (BMI) and without any metabolic abnormalities. Out of a cohort of 1658 middle-aged subjects, representative of the local sanitary districts of the province of Asti (north-western Italy) enrolled for metabolic screening: 241 (14.5%) showed normal BMI, glucose tolerance, blood pressure and waist values and no dyslipidaemia. RESULTS: In this subgroup of subjects, those with hs-CRP levels > or = 3 mg L(-1) showed significantly higher median insulin and HOMA-IR values (respectively: 20.4 vs. 6.0 pmol L(-1), and 0.8 vs. 0.2 microU mL(-1)x mmol L(-1)). In a multiple regression model, insulin and insulin resistance remained significantly and independently related to hs-CRP levels, after adjustments for age, sex, BMI, waist, alcohol consumption, level of physical activity and smoking habits. Very few individuals within lower fasting insulin quartiles showed hs-CRP values > or = 3 mg L(-1) when compared with approximately 60% of those within the highest quartile. CONCLUSIONS: The novel finding is that a state of low-grade systemic inflammation is present in normal BMI subjects who show subclinical insulin resistance but no other metabolic abnormalities.

Prior gestational hyperglycemia: a long-term predictor of the metabolic syndrome.

Little is known about the association between prior gestational hyperglycemia of different severity and the subsequent risk for the metabolic syndrome. Eighty-one women with prior gestational diabetes mellitus (GDM), 25 with one abnormal value at the oral glucose tolerance test (OGTT), and 65 with normal OGTT were studied after a mean of 8.5 yr from the index pregnancy. Patients with prior gestational hyperglycemia (both one abnormal value at the OGTT and GDM) showed a worse metabolic pattern than subjects with gestational normoglycemia [respectively higher values of body mass index (BMI), waist, blood pressure, serum glucose, insulin, C-peptide, homeostatic model assessment (HOMA), fibrinogen and lower levels of HDL-cholesterol]. Prevalence of the metabolic syndrome and its components was 2-4-fold higher in women with prior gestational hyperglycemia (and 10-fold higher if pre-pregnancy obesity coexisted) when compared to normoglycemic controls; in a Cox proportional hazard model, after adjustments for age and pre-pregnancy BMI, gestational hyperglycemia and pre-pregnancy BMI predicted subsequent metabolic syndrome [respectively: hazard ratio (HR)=4.26 and HR=1.21] and most of its components. In the same model, the highest quartile of fasting serum glucose at the OGTT of the index pregnancy was significantly associated to the metabolic syndrome and its components. Gestational hyperglycemia and fasting glucose values were also associated to subsequent fibrinogen values, but not to albumin excretion rates. In young adult women, prior gestational hyperglycemia (particularly abnormal fasting glucose values), above all in combination with pre-pregnancy obesity, anticipates a subsequent syndrome at high cardiovascular risk and, possibly, a mild chronic inflammatory response.

In vivo oxidizability of LDL in type 2 diabetic patients in good and poor glycemic control.

We aimed to determine if increased non-enzymatic glycosylation of the LDL was sufficient to increase the susceptibility to in vivo oxidation of the LDL particles. Twenty-two type 2 diabetic patients (11 males and 11 females) were included in this study. They were enrolled on the basis of good [glycated hemoglobin (HbA1c) < 7%] and poor glycemic control [(HbA1c) > 8%]. LDL were isolated by sequential ultracentrifugation and analyzed by capillary electrophoresis (CE) for diene conjugate content and for electronegativity. The glyc-LDL levels were increased in all diabetic type 2 patients, peaking in the diabetic subjects in poor diabetic control (17.3 +/- 8.07%). The LDL content of diene conjugates was similar between the two groups (6.65 +/- 0.77% for the patients with good glycemic control versus 6.88 +/- 0.74% for those with poor glycemic control; P = 0.49) as was the electrophoretic mobility ((-1.14544 +/- 0.089) x 10(-4) cm2/(V s) for the patients with good glycemic control and (-1.13666 +/- 0.073) x 10(-4) cm2/(V s) for those with poor glycemic control; P = 0.80). The susceptibility to in vivo oxidation of LDL from type 2 diabetic patients in poor glycemic control did not differ from that of well-controlled diabetic patients. LDL glycosylation was not able to increase the oxidizability of LDL in the diabetic patients with poor glycemic control.