Benjamin Uberti

Tamoxifen as a new therapeutic tool for neutrophilic lung inflammation.

Neutrophilic asthma is an important disease subgroup, including patients with severe phenotypes and erratic responses to standard treatments. Tamoxifen (TX), a selective estrogen receptor modulator (SERM) used as treatment of human breast cancer, has been shown to induce early apoptosis of equine blood and bronchoalveolar lavage fluid (BALF) neutrophils in vitro. Equine recurrent airway obstruction (RAO) is a naturally occurring neutrophilic condition, closely related with human asthma. Our purpose was to investigate the therapeutic potential of tamoxifen in horses with neutrophilic lung inflammation.

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.

Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses

The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adult horses without visible wounds or gait alterations were selected. Spinal cord segments were obtained post-mortem for immunohistochemical and immunocolocalization assays. Immunodetection of spinal cord dorsal horn astrocytes was done using a polyclonal goat antibody raised against Glial Fibrillary Acidic Protein (GFAP) and a polyclonal rabbit antibody against Connexin 43 (Cx-43). For immunodetection of spinal cord dorsal horn microglia, a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Epifluorescence and confocal images were obtained for the morphological and organizational analysis. Evaluation of shape, area, cell diameter, cell process length and thickness was performed on dorsal horn microglia and astrocyte. Morphologically, an amoeboid spherical shape with a mean cell area of 92.4 + 34 µm2 (in lamina I, II and III) was found in horse microglial cells, located primarily in laminae I, II and III. Astrocyte primary stem branches (and cellular bodies to a much lesser extent) are mainly detected using GFAP. Thus, double GFAP/Cx-43 immunolabeling was needed in order to accurately characterize the morphology, dimension and cell density of astrocytes in horses. Horse and rodent astrocytes seem to have similar dimensions and localization. Horse astrocyte cells have an average diameter of 56 + 14 µm, with a main process length of 28 + 8 µm, and thickness of 1.4 + 0.3 µm, mainly situated in laminae I, II and III. Additionally, a close association between end-point astrocyte processes and microglial cell bodies was found. These results are the first characterization of cell morphology and organizational aspects of horse spinal glia. Iba-1 and GFAP/Cx-43 can successfully immune-label microglia and astrocytes respectively in horse spinal cords, and thus reveal cell morphology and corresponding distribution within the dorsal horn laminae of healthy horses. The conventional hyper-ramified shape that is normally visible in resting microglial cells was not found in horses. Instead, horse microglial cells had an amoeboid spherical shape. Horse protoplasmic astroglia is significantly smaller and structurally less complex than human astrocytes, with fewer main GFAP processes. Instead, horse astrocytes tend to be similar to those found in rodent’s model, with small somas and large cell processes. Microglia and astrocytes were found in the more superficial regions of the dorsal horn, similarly to that previously observed in humans and rodents. Further studies are needed to demonstrate the molecular mechanisms involved in the neuron-glia interaction in horses.

Modulatory role of regulatory T cells in a murine model of severe equine asthma

BackgroundIt is accepted that T regulatory cells (Treg) control different types of immune responses. In connection with this role, we have recently described an important increase in CD4+, CD25high, Foxp3+ lymphocytes in the airway system of horses coursing with an exacerbation of severe equine asthma (EA). To explore the potential role of this population in the resolution of EA inflammation, we used a murine experimental model in which airway neutrophilic inflammation, which is similar to that observed in EA, is induced in mice by continual exposure to Aspergillus fumigatus contaminated hay. This model has the advantage that in mice we may induce a reduction of the Treg population using low doses of cyclophosphamide (Cy).ResultsThe results indicated that the percentage of Treg cells increased with allergen exposure, as in horses; and animals partially depleted of Treg cells by treatment with Cy showed increased airway inflammation, demonstrated by an increased percentage of neutrophils and specific immunoglobulins in bronchoalveolar lavage fluid (BALF). Furthermore, a histopathologic study of animals that were pretreated with Cy before antigenic challenge showed higher cellular infiltration in the lung and deeper remodeling changes in the bronchi, including epithelial and goblet cell hyperplasia as well as airway smooth muscle hypertrophy.ConclusionIn this murine model of EA, the reduced number and function of Treg induced by low doses of Cy, which directly correlates with increased airway inflammation and lung infiltration, indicates that Treg may play a major role in the regulation and resolution of EA.

Periurethral Vascular Hamartoma in a 6-Month-Old Foal With Idiopathic Hematuria: New Differential Diagnosis

ABSTRACT Hamartomas are nonmalignant masses of normal tissue organized in a chaotic manner. Here, we describe a 6‐month‐old 120 kg Chilean Criollo foal that manifested chronic hematuria observed since birth. Severe anemia and lack of development were the main complaints of this referral. The foal was depressed and in poor body condition, and had lagged development and a coarse haircoat. Mucous membranes were pale, and numerous blood clots were observed toward the end of micturition. Severe normocytic normochromic anemia was confirmed by hematological analysis; there were no significant findings in serum biochemistry or coagulation tests. Transabdominal ultrasonography and urinary tract endoscopy yielded no clinically relevant results. Empirical treatment was initiated on a tentative diagnosis of idiopathic renal hematuria (dexamethasone 0.1 mg/kg [0.045 mg/lb], q 24 hours, IV, sodium ceftiofur 2.2 mg/kg [0.99 mg/lb], IM, q 24 hours, and blood transfusions), but the foals condition further deteriorated, warranting euthanasia. Necropsy revealed a vascular malformation on the extraluminal portion of the proximal urethra at the bladder junction, with a 3 mm urethral communication. Histopathologic examination confirmed this mass to be a hamartoma of vascular origin, which incidentally communicated with the urethral lumen and led to progressive blood loss. In this case, the location of this malformation impeded its discovery and ultimately an accurate diagnosis. Hamartomas are currently not listed as a differential diagnosis for bleeding‐related urinary tract disorders in the modern literature; therefore, we propose that they should be considered as a differential diagnosis in cases of unexplained or refractory idiopathic hematuria. HighlightsUnexplained chronic hematuria and anemia in a 6‐month‐old Chilean Criollo foal with ill thrift.A vascular malformation communicating with the urethral lumen caused progressive blood loss.This is the first description of a periurethral vascular hamartoma in horses.

Tamoxifen inhibits chemokinesis in equine neutrophils

Neutrophils are terminally differentiated innate effector cells at the first line of host defense. Neutrophil migration within tissues is complex and involves several steps, during which these cells must be able to interpret a variety of chemical and physical signals. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including equine asthma. 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 bronchoalveolar lavage fluid (BALF) neutrophils, reduction of BALF neutrophil content, and improvement in animals’ clinical status. Further, TX dampens chemotactic index and respiratory burst production in vitro. The aim of this study was to provide information on the effect of TX on chemokinesis in peripheral blood neutrophils from five healthy horses. Results showed that neutrophils increased migration and travelled distance in response to IL-8; but in the presence of TX, IL-8 did not produce neutrophil migration. This suggests that TX has an inhibitory effect on the kinesis of equine peripheral blood neutrophils stimulated with IL-8. However, further studies are required to fully understand the signaling pathways of TX on neutrophil chemokinesis.

Microglia and astrocyte activation in the spinal cord of lame horses

OBJECTIVE To determine the microglial and astrocyte response to painful lameness in horses. STUDY DESIGN Ionized calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) expression, cell density and morphology were determined through immunofluorescence within the dorsal horn of equine spinal cord. ANIMALS A total of five adult horses with acute or chronic unilateral lameness, previously scheduled for euthanasia. METHODS Musculoskeletal lameness was evaluated in five horses through visual evaluation according to clinical guidelines. Spinal cord samples were obtained immediately after euthanasia, and distal limb lesions were confirmed through dissection and radiography. Iba-1 immunostaining was used for detection and characterization of dorsal horn microglia. GFAP was used for immunostaining of dorsal horn astrocytes. Iba-1 and GFAP labeled cells were quantified in the dorsal horn, and intensity of fluorescence was compared between the ipsi- and contralateral dorsal horn to the affected limb, and between dorsal horn segments of all horses. RESULTS Iba-1 expression was higher in the ipsilateral dorsal horn of the affected limb in contrast to the contralateral side dorsal horn. GFAP markers did not demonstrate increased astrocytic activity on the dorsal horn ipsilateral side to the distal limb lesion of affected horses. Horses with acute lameness predominantly had a spherical shape microglial phenotype, while cells from chronic lameness cases had variable morphology. Astrocytes evidenced small somas and large processes in both acute and chronic lameness, with higher GFAP localization in the main branches. As in the case of rodents, the localization of microglia and astrocytes in horses was mainly situated within laminae I, II and III. CONCLUSIONS AND CLINICAL RELEVANCE Iba-1 and GFAP are functional and morphological markers of spinal microglial cells and astrocytes in horses with lameness.

Concentration of the CS-846 Epitope in Serum and Synovial Fluid of Horseswith Different Grades of Osteochondral Fragments in the Carpal Joints

The aim of this study was to determine serum and synovial concentrations of the CS-846 epitope of articular cartilage aggrecan, in horses with a radiological diagnosis of osteochondral fragmentation (OF). For this purpose, 20 thoroughbred horses with unilateral radiocarpal or intercarpal OF were used, and 10 clinically and radiologically healthy Thoroughbred horses were assigned to the control group. Serum and synovial concentrations of the CS-846 epitope were measured in both groups by means of ELISA. The concentration of the CS-846 epitope in synovial fluid was significantly higher in carpal joints with moderate articular damage (grade 2 OF) than in the control group or in horses with severe disease (grade 3 and 4 OF). In serum, non-statistical differences in the concentration of the CS-846 epitope were observed between horses with OF and controls. We conclude that the concentration of CS-846 epitope in synovial fluid suggests an increase in cartilage aggrecan synthesis, which may be associated with the presence of moderate clinical disease. The CS-846 epitope concentration in synovial fluid may be a useful biomarker for the study of carpal OF in horses, providing a measure of the balance between cartilage synthesis and degradation in this equine disease.