First Documented Pathologies in Tenontosaurus tilletti with Comments on Infection in Non-Avian Dinosaurs.

In 2001, a nearly complete sub-adult Tenontosaurus tilletti was collected from the Antlers Formation (Aptian-Albian) of southeastern Oklahoma. Beyond its exceptional preservation, computed tomography (CT) and physical examination revealed this specimen has five pathological elements with four of the pathologies a result of trauma. Left pedal phalanx I-1 and left dorsal rib 10 are both fractured with extensive callus formation in the later stages of healing. Left dorsal rib 7 (L7) and right dorsal rib 10 (R10) exhibit impacted fractures compressed 26 mm and 24 mm, respectively. The fracture morphologies in L7 and R10 indicate this animal suffered a strong compressive force coincident with the long axis of the ribs. All three rib pathologies and the pathological left phalanx I-1 are consistent with injuries sustained in a fall. However, it is clear from the healing exhibited by these fractures that this individual survived the fall. In addition to traumatic fractures, left dorsal rib 10 and possibly left phalanx I-1 have a morphology consistent with post-traumatic infection in the form of osteomyelitis. The CT scans of left metacarpal IV revealed the presence of an abscess within the medullary cavity consistent with a subacute form of hematogenous osteomyelitis termed a Brodie abscess. This is only the second reported Brodie abscess in non-avian dinosaurs and the first documented occurrence in herbivorous dinosaurs. The presence of a Brodie abscess, known only in mammalian pathological literature, suggest mammalian descriptors for bone infection may be applicable to non-avian dinosaurs.

Assessment of the anti-c-kit monoclonal antibody YB5.B8 in affinity magnetic enrichment of human lung mast cells.

The monoclonal antibody, YB5.B8 binds to the second domain of the c-kit proto-oncogene product on human mast cells, a receptor associated with tyrosine kinase activity. This molecule is involved with cell proliferation, maturation and viability as well as cell activation and its natural ligand is stem cell factor (SCF). We have used this antibody coupled to Dynabeads to perform positive affinity enrichment of human lung mast cells. This procedure results in enrichment of mast cells from 2.6 +/- 0.3% to 85.0 +/- 1.6% purity (n = 29) with yields of 41.9 +/- 3.7% (n = 29). As YB5.B8 interacts with the same receptor domain as does SCF, it is important to demonstrate that this procedure does not modify mast cell function. Incubation of mast cells with 1-5000 ng/ml YB5.B8 for 30 min neither induced histamine release nor modulated histamine release induced by anti-IgE. Furthermore, incubation with YB5.B8 did not alter prolonged culture with SCF. Examination of cells enriched using YB5.B8 showed that they had a normal histamine content (3.8 +/- 0.3 pg/cell compared with 3.9 +/- 0.7 pg/cell unpurified, n = 20) and had unchanged behaviour in both histamine secretion and cell survival studies. These studies indicate that YB5.B8 does not influence mast cell function and thus its use in magnetic affinity purification procedures offers a simple and effective method for enriching human mast cell preparations.

Monoclonal antibodies specific for guinea pig eosinophil major basic protein: their use in an ELISA, immunocytochemistry and flow cytometry.

Eosinophil major basic protein (MBP), purified from guinea pig eosinophil granules was used to raise five monoclonal antibodies (MoAbs). Their reactivity with MBP was confirmed by immunoblotting and indirect ELISA. Two of the MoAbs were used to develop a sensitive and specific antigen capture (sandwich) ELISA for guinea pig eosinophil MBP which gives an accurate and reproducible standard curve over the range of 10-10,000 ng/ml. The specificity of the ELISA for MBP was confirmed and its suitability for testing biological samples ascertained by measurement of MBP in bronchoalveolar lavage fluid (BALF) and plasma from guinea pigs sensitized and challenged with ovalbumin. The ELISA was also capable of detecting MBP in culture supernatants from purified eosinophil preparations challenged with calcium ionophore in vitro. One of the monoclonals could be used to strongly and specifically stain guinea pig eosinophils in immunocytochemistry, whilst all five could be used to visualize eosinophils in suspension in BALF or peritoneal lavage fluid by flow cytometry. There was no staining of other guinea pig leucocyte types, nor crossreactivity with human eosinophils by immunocytochemistry or flow cytometry.

Interleukin 4 is localized to and released by human mast cells.

Recent attention has focused on the T helper type 2 (Th2) lymphocyte as a source of interleukin 4 (IL-4) in allergic disease. However, Th2 cells themselves require a pulse of IL-4 to initiate this synthesis. Here we provide immunohistochemical evidence of IL-4 localization to human mast cells of the skin and respiratory tract, and demonstrate that immunoglobulin E-dependent stimulation of purified human lung mast cells leads to the rapid release of IL-4 into the extracellular environment. We propose that mast cell activation in an allergic response provides a rapid and local pulse of IL-4 into the local environment essential for the triggering of T lymphocytes into sustained IL-4 production and to initiate inflammatory cell accumulation and activation.

Density profile of bronchoalveolar lavage eosinophils in the guinea pig model of allergen-induced late-phase allergic responses.

Inhalation of aerosolized ovalbumin by guinea pigs both during sensitization and upon challenge induces a pulmonary eosinophilia as assessed by cells recovered in bronchoalveolar lavage fluid (BALF). In comparison with BALF eosinophil numbers in naive animals of 0.82 +/- 0.2 x 10(6) cells, those in sensitized animals before challenge and 17 and 72 h after challenge were 1.48 +/- 0.2 x 10(6), 2.60 +/- 0.6 x 10(6), and 4.2 +/- 0.7 x 10(6) cells, respectively. BALF eosinophils from all these groups were notable for their heterogeneity with respect to density, size, and appearance under the electron microscope. In comparison with peritoneal eosinophils, which had a single mean density peak of 1.088 +/- 0.001 g/ml, BALF cells comprised hypodense (less than 1.080 g/ml), normodense (1.080 to 1.096 g/ml), and hyperdense (greater than 1.096 g/ml) eosinophils. The percentage of hypodense eosinophils rose from 25% in naive animals to 63% in sensitized animals (P less than 0.001) and fell after challenge. In contrast, challenge induced the appearance of hyperdense eosinophils, which rose from 6% in sensitized animals to 42% 72 h after challenge (P less than 0.001). Blood eosinophils in naive animals showed a similar profile to those in the lung, but after sensitization and challenge no gross changes in the proportion of either hypodense or hyperdense eosinophils were observed. Flow cytometric analysis of BALF eosinophils indicated that hypodense eosinophils, with a mean diameter of 15.8 microns, were larger than both normodense and hyperdense eosinophils, which had mean diameters of 14.3 and 11.6 microns, respectively. Although the numbers and size of granules were not reduced in hypodense BAL eosinophils, electron microscopy morphology indicated a reduced granular content.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and secretory characteristics of bovine lung and skin mast cells: evidence for the existence of heterogeneity.

We have examined cells dispersed enzymatically from three different sites in the bovine lung (tracheal mucosa, bronchial mucosa and parenchyma) and the skin, in order to ascertain whether the bovine model could be used to study mast cell heterogeneity. Histochemically there were two sub-populations of mast cells present in both lung and skin (on the basis of toluidine blue staining and the sensitivity to formalin fixation), but their proportions were similar in all sites studied. Skin mast cells contained approximately twice the amount of histamine than their counterparts in the lung (P less than 0.05). Functional heterogeneity was examined by in vitro release of histamine following secretagogue challenge. Calcium ionophore induced a substantial release of histamine; skin mast cells releasing significantly more histamine than any of the lung mast cells (at 10 microM ionophore, 37.1% and 20.7% net histamine release, respectively, P less than 0.05), although the time-course of release from the two tissues was similar. The neuropeptides vasoactive intestinal peptide and somatostatin induced a modest but statistically significant release of histamine from both skin and lung mast cells, whilst substance P only induced histamine secretion from skin mast cells. A range of other potential immunological and non-immunological secretagogues was unsuccessful in eliciting histamine release from mast cells in any of the tissues. We conclude that there were no convincing histochemical differences between mast cells from the sites examined in the lung or skin. Additionally, there was no discernable functional heterogeneity between mast cells within the lung, but functional differences were evident between mast cells of the bovine lung and skin. However, in the absence of a suitable immunological stimulus the bovine model cannot be regarded as a good model of mast cell heterogeneity.

Leukotriene B4 induces enhanced migration of fish leucocytes in vitro.

Leukotriene B4 (LTB4) was found to induce enhanced migration of the eosinophilic G1 granulocyte of the dogfish Scyliorhinus canicula in the migration under agarose assay. Higher levels of LTB4, however, were required to produce this effect than with mammalian neutrophils under similar conditions. It is postulated that this may be due to the dogfish granulocytes possessing fewer receptors for LTB4 than their mammalian counterparts. The eosinophilic G3 granulocyte was also tested using the same assay but results were inconclusive. The effect of LTB4 on dogfish G1 and G3 granulocytes was also monitored with the bipolar shape formation (BSF) assay. LTB4 induced BSF in both granulocyte types, and this method appeared to be more sensitive than the migration under agarose assay. Whether the enhanced migration observed is a result of chemotaxis or chemokinesis is not determined. This present study represents the first known report of the function of LTB4 in a non-mammalian vertebrate.

Preliminary studies on the chemotactic potential of dogfish (Scyliorhinus canicula) leucocytes using the bipolar shape formation assay.

The bipolar shape formation assay, previously used to determine the chemotactic potential of various factors for mammalian leucocytes, was tested in the present study with granulocytes of the lesser spotted dogfish, Scyliorhinus canicula. Bipolar shape formation was found to be a temperature dependent process with maximal formation observed at 30 degrees C. Addition of the formyl peptide, N-formyl-methionyl-phenylalanine failed to induce any bipolar forms at all temperatures and concentrations tested.