Effects of anti-inflammatory drugs on increased vascular permeability in acute inflammatory response in the chicken.

The effects of anti-inflammatory drugs were examined on the turpentine-induced acute inflammatory response in the chicken skin. An increase in vascular permeability was evaluated using the 'dye' technique. Although histamine and 5-hydroxytryptamine (5-HT) appeared to be the major mediators of the permeability response in the early phase, involvement of 5-HT was more significant. Use of other anti-inflammatory drugs suggested mediation of the permeability response also through the release of prostaglandins or leukotrienes, or both.

The avian inflammatory-reparative response: adaptation and utilization of a tissue cage model.

A tissue cage model was adapted and utilized for examination of the inflammatory-reparative response in the chicken. The model involved insertion of a polyethylene cage, soaked in the irritant, into the previously prepared subcutaneous pouch. Using this model the cellular events in the fibrovascular granulation tissue in response to turpentine, concanavalin-A and Escherichia coli endotoxin were studied. It was concluded that the tissue cage model worked well in the chicken and that it can be useful in studies relating to the inflammatory-reparative response.

Studies on the cellular response in avian inflammation using a simple subcutaneous pouch model.

A subcutaneous pouch model was developed and used for examination of cellular events in the local acute inflammatory response in the chicken. The model involved insertion of a pair of coverslips-dipped in the irritant-into the prepared subcutaneous pouch, and their replacement with plain coverslips at frequent intervals. The model was simple to operate, caused minimal distress to the experimental bird, and permitted multiple continual sampling of emigrating cells. The cytological features and migratory sequence of inflammatory cells produced by non-immunological and immunological stimuli were studied over a period of 48 h. The initial migration of leukocytes comprised heterophils and monocytes. This was soon followed by emigration of basophils. The basophilic reaction was pronounced in response to Escherichia coli endotoxin. Lymphocytes were prominent in the later stages. Also, in the later stages the monocytes apparently coalesced, formed syncytia and even what appeared to be distinct multinucleated giant cells. The results obtained by the present model corroborated the findings of earlier investigators recorded through the use of tissue sections and impression smears. It is suggested that the subcutaneous pouch model may offer an excellent system in avian inflammation research.

Pathology of inflammatory-reparative response in punched wounds of the chicken skin.

Vascular and cellular responses were examined sequentially in punched wounds of the chicken skin. The 'dye' and the 'colloidal carbon' technique revealed that the increased permeability response was immediate and sustained. The response attained its peak at 1 hour and persisted for up to 6 hours, but the increased permeability was prolonged over a period of 48 h. The increase in vascular permeability at all stages was confined to venules and small veins only. In the later stages of healing, the newly formed vessels in the granulation tissue were leaky. The initial migration of leukocytes comprised heterophils and monocytoid cells. This was soon followed by infiltration of some basophils. A striking feature of the reaction, in the granulation tissue, was that the monocytoid cells apparently coalesced, formed syncytia, and even what appeared to be distinct multinucleated giant cells. Whereas mast cells participated in the early stages, they were absent in the granulation tissue. Proliferation of the fibroblasts began around 18 h, angiogenesis and re-epithelialization at 3 days, and the healing was complete by about 7 days.

Increased vascular permeability and leucocyte emigration in Escherichia coli endotoxin injury in the chicken skin.

The permeability response was examined in chickens following intradermal injection of Escherichia coli endotoxin. The 'dye' and 'colloidal carbon' techniques were employed. The endotoxin evoked a monophasic response of immediate-prolonged type. The increase in vascular permeability was confined to venules and small veins only, indicating its mediation by endogenous permeability factors. The carbon labelling exhibited arboreal, disjointed and rectangular to hexagonal patterns. Histologically, a striking feature of the reaction was an accumulation of basophils in unusually large numbers. No other type of stimulus appears to induce basophilic response of a similar magnitude in the chicken. The results suggest that endotoxin, being a bacterial product, may exert a chemotactic effect on basophils. Hyperaemia, oedema, necrosis and formation of perivascular lymphoid aggregates were also recognised.

Detection of anti-equine neutrophil antibody by use of flow cytometry.

Flow cytometric and conventional fluorescence microscopic methods were compared to detect heterologous (rabbit) neutrophil antibody bound to equine neutrophils. Unfixed and paraformaldehyde-fixed neutrophils were treated with normal rabbit serum or various dilutions of an antineutrophil serum. The cells were then reacted with fluorescein conjugates of goat anti-rabbit IgG, staphylococcal protein A, and streptococcal protein G. Antibody binding was evaluated by use of fluorescence microscopy and flow cytometry. Unfixed neutrophils treated with normal rabbit serum did not fluoresce, whereas many of the fixed neutrophils had distinct cytoplasmic and some membranous (nonspecific) fluorescence. Unfixed cells treated with the antiserum had localized areas (capping) of intense membrane fluorescence, whereas fixed cells had bright uniform membranous fluorescence. The intensity of specific fluorescence varied with the antiserum dilution and the conjugate. On flow cytometry, over 80% of unfixed cells treated with antiserum dilutions up to 1:1,024, 1:2,048, and 1:256 fluoresced, respectively, with anti-IgG, protein-G, and protein-A conjugates. Fixed cells generally had similar percentages of fluorescent cells, but at a higher (1-step) antiserum dilution. It was concluded that flow cytometry is more sensitive than conventional fluorescence microscopy to detect antibodies associated with equine neutrophils.

Detection of equine antiplatelet and antineutrophil antibodies by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was standardised and applied for the detection of antiplatelet and antineutrophil antibodies using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. The standardised technique consisted of using Immulon type 3 plate, 1 per cent gelatine as a blocking solution, poly-L-lysine buffer as a coating solution, unfixed antigen, 90 microliters test serum, horseradish peroxidase conjugated antibody and o-phenylenediamine dihydrochloride as a substrate. The number of unfixed platelets or neutrophils required for optimum detection of antibodies was 250,000 per well. Unfixed cellular antigens were as good as their extracts and superior to paraformaldehyde-fixed antigens in detecting specific antibodies. Microtitre plates coated with platelet or neutrophil antigens could be stored at 4 degrees and -70 degrees C for four to five weeks without significant loss of antigenicity. The ELISA was very sensitive in that antiplatelet antibody was detected up to a titre of 1:204,800 and antineutrophil antibody to a titre of 1:51,200. Some cross-reactivity (1:1600) was detected in antiplatelet and antineutrophil sera for neutrophil and platelet antigens, respectively. Platelet-associated antibody was also detected in extracts from platelets pretreated with 1:2 and 1:8 dilutions of antiplatelet serum. Standardised ELISA detected antiplatelet antibodies in nine and antineutrophil antibodies in three of 100 isologous equine blood typing sera.

Ultrastructural and biochemical observations on antineutrophil antibody- and complement-induced immuno-injury to equine neutrophils.

Antibody-induced damage to neutrophils was studied to elucidate processes associated with destruction of neutrophils in immune-mediated neutropenias. Cytomorphological changes and release of certain cellular constituents were determined for neutrophils treated with an antineutrophil serum in the presence or absence of rabbit complement. Neutrophils exposed to the antineutrophil serum alone showed endocytotic vacuoles and degranulation. In contrast, neutrophils exposed to the antineutrophil serum and complement showed marked morphologic changes. The plasma membrane developed numerous vesicles, villous processes and minute areas of bilayer discontinuity. Highly damaged cells exhibited cellular and nuclear swellings, disruption of cytoplasmic integrity and disordered distribution of lysosomal granules. Cytoplasmic constituents (K+ and lactate dehydrogenase) were released extracellularly from neutrophils exposed to the antineutrophil serum with or without complement. Cytological changes induced by the antineutrophil serum and complement were analogous to those reported for leucocytes exposed to the activated complement components C5b-9 (the membrane attack complex) and bacterial toxins. It was concluded that the cytological abnormalities observed were most probably associated with immune-mediated damage to the cell membrane, leading to leakage of cytoplasmic constituents like K+, colloidal osmotic swelling, and disruption of the cytoskeletal system.

Detection of antiplatelet antibody: comparison of platelet immunofluorescence, agglutination, and immunoinjury tests using rabbit antiequine platelet serum.

Immunofluorescence, tube agglutination, and platelet factor-3 immunoinjury tests for detecting antiplatelet antibody were compared using a heterologous system of equine platelets and rabbit antiequine platelet serum. Platelet immunofluorescence tests were performed using paratormaldehyde-fixed platelets in suspension as well as in air-dried smears on glass slides (solid phase). Bright homogeneous, membranous, specific fluorescence was seen in both assays with anti-immunoglobulin G (IgG) and protein G fluorescein isothiocynate conjugates (FITC-conjugates). Protein A conjugate gave nonspecific fluorescence irrespective of normal or antiserum treatment. Anti-IgG and protein G conjugates in suspension immunofluorescence tests with the same antiserum yielded antibody titers of 1:1024 and 1:128, respectively. Similarly, respective titers of 1:512 and 1:64 were obtained with solid phase immunoassay. Platelet suspension assay was slightly better than the solid phase assay. These observations indicated that anti-IgG was more sensitive than protein G in detecting antiplatelet antibody by fluorescence microscopy, while protein A was ineffective because of its nonspecificity. Chloroquine treatment of platelets failed to reduce the nonspecific fluorescence. Platelet agglutination and platelet factor-3 tests were relatively less sensitive to detect equine antiplatelet antibody.

Methods for detection of immune-mediated neutropenia in horses, using antineutrophil serum of rabbit origin.

Equine neutrophil antibody was raised in rabbits inoculated with equine neutrophils isolated to purity greater than 99.0%, using Percoll density-gradient sedimentation. Neutrophil antibody was detected by use of agar gel diffusion, leukoagglutination, indirect immunofluorescence, staphylococcal protein A and streptococcal protein G binding, and phagocytic inhibition techniques. Precipitin lines and leukoagglutination were seen in antiserum dilutions of 1:4 and 1:64, respectively. The specific nature of leukoagglutination was characterized by the formation of rosette-like clumps of neutrophils. Specific bright membranous fluorescence was seen in neutrophils treated with the antiserum and exposed to fluorescein-conjugated goat anti-rabbit immunoglobulin, and staphylococcal protein A and streptococcal protein G. Whereas the indirect immunofluorescence and protein G-binding tests were equally sensitive and resulted in titer of 1:256, the protein A-binding test was less sensitive and resulted in titer of only 1:32. Nonspecific binding of protein A and protein G was noticed as uniform or patchy cellular fluorescence in a small number of neutrophils. Treatment of neutrophils with antiserum up to dilution of 1:8 resulted in a significant (P less than 0.05) suppression of phagocytosis of opsonized zymosan particles. Thus, protein G-binding and indirect immunofluorescence tests are highly sensitive to detect neutrophil antibody and may be used to diagnose immune-mediated neutropenias in horses and, possibly, in other animal species.

Haematological changes in buffalo calves inoculated with Escherichia coli endotoxin and corticosteroids.

Haematological studies were conducted on 10 clinically normal water buffalo calves to determine leucocytic responses to Escherichia coli endotoxin, prednisolone and dexamethasone. Intravenous injection of 10 micrograms endotoxin induced minimal decreases in leucocyte numbers, whereas 20, 50 and 100 micrograms produced a marked leucopenia within one hour. Moderate to marked leucopenia, neutropenia and lymphopenia persisted for three to 14 hours. Significant rebound neutrophilia was evident at six to eight hours after inoculation in calves given only 10 and 20 micrograms. Intramuscular injection of prednisolone (100 mg) and dexamethasone (5 mg) produced increases in total leucocyte counts and neutrophil numbers within two hours. Moderate to marked leucocytosis and neutrophilia persisted for eight to 24 hours. Lymphocyte response was unlike that in other species in that lymphopenia was not a consistent feature of the corticosteroid response. A transient monocytosis was seen following administration of prednisolone but not of dexamethasone, while eosinopenia and basopenia developed in both cases. In conclusion, endotoxin and corticosteroid induced changes in total and differential leucocyte counts in water buffalo were largely similar to those seen in cattle.

Ventricular intussusception in a domestic fowl.

Necropsy of a 12-week-old female chicken revealed invagination of proventriculus into the gizzard with eversion of the former.

Pathology of passive mesenteric anaphylaxis in the chicken.

Passive mesenteric anaphylaxis (PMA) was produced on intravenous challenge by bovine serum albumin in chickens which had received two hours earlier an intraperitoneal injection of anti-BSA chicken serum. Sequential study of permeability and cellular responses, using colloidal carbon, revealed a biphasic pattern of increased permeability which was confined to venules and small veins only. The cell population comprised an infiltration of heterophils, monocytes and basophils. Marked degranulation and disruption of mast cells accompanied by a significant decrease in their number suggested their active participation. There was strong evidence that the activated mediator cells were both mast cells and basophils. It is suggested that PMA may offer an excellent experimental model in avian anaphylaxis research.

A microscopic study of increased vascular permeability and leucocyte emigration in passive cutaneous anaphylaxis in the chicken.

Passive cutaneous anaphylaxis was produced in chickens with bovine serum albumin (BSA) and anti-BSA chicken serum. Colloidal carbon was then given intravenously to identify the leaky vessels. Examination of the resulting sequential changes revealed a marked increase in vascular permeability affecting the venules. A noteworthy feature of the reaction was the early participation of the basophils, which contained phagocytosed carbon particles. Eosinophils were absent.

Quantitative estimation of increased vascular permeability in acute inflammatory reaction in the chicken skin.

An increase in vascular permeability was estimated quantitatively in acute inflammatory reaction in the chicken using passive cutaneous anaphylaxis as an experimental model. Dye exuded in the cutaneous lesion was extracted by formamide and measured spectrophotometrically. The technique, though time-consuming, worked well in the chicken. The results suggested that the method can be profitably utilised in studies relating to avian inflammation.

Suppression by mepyramine maleate of the increased vascular permeability in passive cutaneous anaphylaxis in the chicken.

Passive cutaneous anaphylaxis was produced in chickens pretreated with the antihistamine mepyramine maleate. Quantitative estimation of the increased vascular permeability in the lesion revealed 79.5% suppression indicating its mediation largely by histamine. The findings suggest that the quantitative estimation of the increased vascular permeability, though time-consuming, is more precise than the visual assessment.