Simultaneous flow cytometric analysis of phagocytosis and oxidative burst activity in equine leukocytes.

This paper describes a method for simultaneously measuring phagocytosis and oxidative burst activity in equine peripheral blood leukocytes by flow cytometry. Opsonized propidium iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes and the oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieve optimal activity of phagocytosis and oxidative burst are described. The advantage of the simultaneous technique is that it provides both independent and comparative values for phagocytosis and the oxidative burst, for the detection of impaired mechanisms of microbial destruction. Furthermore, the technique allows evaluation of opsonization activity in this context.

Efficacy of salmeterol xinafoate in horses with recurrent airway obstruction.

OBJECTIVE: To determine the onset, magnitude, and duration of bronchodilation after administration of aerosolized salmeterol xinafoate in horses with recurrent airway obstruction. DESIGN: Randomized controlled study ANIMALS: 6 horses with recurrent airway obstruction. Procedure Horses received aerosolized salmeterol (210 microg) or no treatment, using a crossover design. Salmeterol was administered, using a mask designed for aerosol delivery in horses. Subjective rating of airway obstruction (RAO), maximal change in pleural pressure (deltaPplmax), and pulmonary resistance (RL) were determined at baseline; 5, 15, and 30 minutes; and 1, 2, 4, 6, 8, 10, and 12 hours after administration of salmeterol and in horses that did not receive treatment. RESULTS: The deltaPpl and RL were improved 15 minutes through 6 hours after administration of salmeterol, compared with values obtained from horses receiving no treatment. The RAO was improved 15 minutes through 2 hours after administration of salmeterol. The maximal response to salmeterol was evident 30 to 60 minutes after administration and was characterized by a 59 + 19% decrease in deltaPpl and a 56 +/- 13% decrease in RL. The deltaPpl and RL were not different from baseline values 8 hours after salmeterol administration. CONCLUSIONS AND CLINICAL RELEVANCE: Duration of action of salmeterol in these horses was approximately 6 hours. Maximal bronchodilation was somewhat delayed (30 to 60 minutes), and the magnitude of response was similar to that of short-acting beta2-adrenergic agonists. Salmeterol provides moderately sustained bronchodilation in horses with recurrent airway obstruction and may be an effective drug for long-term control of this condition.

Determination of synovial fluid and serum concentrations, and morphologic effects of intraarticular ceftiofur sodium in horses.

OBJECTIVES: To determine the serum and synovial fluid concentrations of ceftiofur sodium after intraarticular (IA) and intravenous (IV) administration and to evaluate the morphologic changes after intraarticular ceftiofur sodium administration. STUDY DESIGN: Strip plot design for the ceftiofur sodium serum and synovial fluid concentrations and a split plot design for the cytologic and histopathologic evaluation. ANIMALS: Six healthy adult horses without lameness. METHODS: Stage 1: Ceftiofur sodium (2.2 mg/kg) was administered IV. Stage 2: 150 mg (3 mL) of ceftiofur sodium (pHavg 6.57) was administered IA into 1 antebrachiocarpal joint. The ceftiofur sodium was reconstituted with sterile sodium chloride solution (pH 6.35). The contralateral joint was injected with 3 mL of 0.9% sterile sodium chloride solution (pH 6.35). Serum and synovial fluid samples were obtained from each horse during each stage. For a given stage, each type of sample (serum or synovial fluid) was collected once before injection and 12 times after injection over a 24-hour period. All horses were killed at 24 hours, and microscopic evaluation of the cartilage and synovium was performed. Serum and synovial fluid concentrations of ceftiofur sodium were measured by using a microbiologic assay, and pharmacokinetic variables were calculated. Synovial fluid was collected from the active joints treated during stage 2 at preinjection and postinjection hours (PIH) 0 (taken immediately after injection of either the ceftiofur sodium or sodium chloride), 12, and 24, and evaluated for differential cellular counts, pH, total protein concentration, and mucin precipitate quality. RESULTS: Concentrations of ceftiofur in synovial fluid after IA administration were significantly higher (P = .0001) than synovial fluid concentrations obtained after IV administration. Mean peak synovial fluid concentrations of ceftiofur after IA and IV administration were 5825.08 microg/mL at PIH .25 and 7.31 microg/mL at PIH 4, respectively. Mean synovial fluid ceftiofur concentrations at PIH 24 after IA and IV administration were 4.94 microg/mL and .12 microg/mL, respectively. Cytologic characteristics of synovial fluid after IA administration did not differ from cytologic characteristics after IA saline solution administration. White blood cell counts after IA ceftiofur administration were < or =3,400 cells/ML. The mean synovial pH of ceftiofur treated and control joints was 7.32 (range, 7.08-7.5) and 7.37 (range, 7.31-7.42), respectively. Grossly, there were minimal changes in synovium or cartilage, and no microscopic differences were detected (P = .5147) between ceftiofur-treated joints and saline-treated joints. The synovial half-life of ceftiofur sodium after IA administration joint was 5.1 hours. CONCLUSIONS: Synovial concentrations after intraarticular administration of 150 mg of ceftiofur sodium remained elevated above minimal inhibitory concentration (MIC90) over 24 hours. After 2.2 mg/kg IV, the synovial fluid ceftiofur concentration remained above MIC no longer than 8 hours. CLINICAL RELEVANCE: Ceftiofur sodium may be an acceptable broad spectrum antimicrobial to administer IA in septic arthritic equine joints.

Pulmonary function and adrenal gland suppression with incremental doses of aerosolized beclomethasone dipropionate in horses with recurrent airway obstruction.

OBJECTIVE: To evaluate clinical response, pulmonary function, and adrenal gland response to incremental doses of beclomethasone dipropionate in horses with recurrent airway obstruction. DESIGN: Crossover trial. ANIMALS: 8 horses with recurrent airway obstruction. PROCEDURE: Horses randomly assigned to 4 groups were treated twice daily via aerosol administration of placebo or 500, 1,000, or 1,500 micrograms of beclomethasone dipropionate in a crossover design with a 10-day minimum washout period. Subjective assessment of airway obstruction, serum cortisol concentration, and maximum change in pleural pressure during tidal breathing (delta Pplmax) were determined daily prior to morning drug administration, and delta Pplmax was reevaluated 15 minutes after morning drug administration. Pulmonary resistance and dynamic compliance were determined at baseline and approximately 12 hours after the final treatment. RESULTS: An immediate treatment effect was not identified. Within 24 hours, delta Pplmax and airway obstruction were lower in horses receiving beclomethasone. Onset and magnitude of response was similar among the 3 beclomethasone dose regimens. Pulmonary resistance was improved only after administration of all 3 doses of beclomethasone, whereas dynamic compliance was improved after administration of 1,000 micrograms and 1,500 micrograms of beclomethasone. Reduction in serum cortisol concentration occurred with all 3 beclomethasone dose regimens; however, the magnitude of adrenal gland suppression was greater in horses receiving 1,000 or 1,500 micrograms of beclomethasone. CONCLUSIONS AND CLINICAL RELEVANCE: Low-dose (500 micrograms) beclomethasone administration caused similar, improvement in pulmonary function, compared with high-dose beclomethasone (1,000 and 1,500 micrograms), with the exception of dynamic compliance, and caused less suppression of endogenous cortisol production.

Pythiosis with bone lesions in a pregnant mare.

A 9-year-old pregnant mare was referred for evaluation of a nonhealing wound of 8 weeks' duration on the lateral aspect of the left forelimb. A soft tissue mass encircled the proximal two thirds of the metacarpus; radiography revealed a moderate periosteal reaction affecting metacarpal bone i.v. Histologic and immunohistochemical examinations revealed eosinophilic granulomatous inflammation and Pythium sp in the soft tissues. The mare was treated for 12 days with antimicrobials, medicated wound dressings, debridement, and i.v. administration of sodium iodide; radiography revealed progression of the bone lesions. The mare was treated by regional arterial perfusion with miconazole and excision of affected soft tissues and the distal two thirds of metacarpal bone i.v. The mare recovered without complications and gave birth to a healthy foal. Regional perfusion of antifungal agents provides high concentrations in soft and osseous tissues and permits use of low dosages of agents administered by other routes, which reduces cost, adverse effects, and teratogenic effects.

Immunomodulation in horses.

Nonspecific immunomodulators are substances that induce non-antigen-specific enhancement of the body's native or acquired defense mechanisms. Immunomodulant preparations are most often used for treatment of chronic, viral, or bacterial infection with evidence of secondary immunosuppression. The proposed mechanism of action of these products is macrophage activation and subsequent release of cytokines that enhance humoral and cell-mediated immunity. In equine medicine, nonspecific immunostimulant products are used for prevention and treatment of infectious respiratory disease and treatment of sarcoid skin tumors.

Characterization of peripheral blood and pulmonary leukocyte function in healthy foals.

Studies in infants and foals indicate an age-dependent maturation of peripheral lymphocyte subsets. The age-dependent relationship for maturation of cellular immune responses, such as phagocytosis and lymphocyte responses of the peripheral and pulmonary-derived leukocytes, has not been characterized in foals. Lymphocyte subpopulations, mitogen stimulation response of lymphocytes, lymphokine-activated killing cell activity, phagocytosis and oxidative burst activity, and serum immunoglobulin (Ig) classes G and M concentrations were determined in developing foals. This study illustrates age-dependent changes in immunoglobulin class concentrations, lymphocyte subsets, and EqMHC Class II expression in cells of the peripheral blood and lungs of developing neonatal-to-weanling foals. The increase in peripheral blood and BAL B-lymphocytes and serum immunoglobulins in developing foals suggests expansion of immune cell populations during a time in which environmental pathogen exposure is great. General immune function, mitogenic responses, LAK cell activity, opsonized phagocytosis, and oxidative burst activity of newborns was similar to the adult horse. Total immune-cell numbers, rather than function, seemed to be the limiting factor in the development of the equine neonatal immune system. There was an age-related percent increase in the appearance of pulmonary lymphocytes, but a percent decrease in macrophages. Although development of the respiratory immune system follows changes in the peripheral blood, cellular expansion, activation, and migration may occur at a slower pace, making the respiratory environment susceptible to pathogens prior to optimal immune system maturity.

Black walnut induced laminitis.

A 5-y-old Paint horse gelding was evaluated for acute laminitis after exposure to black walnut shavings. The gelding's feet were previously soaked in an ice bath continuously for approximately 24 h. Treatment consisted of anti-inflammatory and vasodilator therapy. Serial radiographs revealed progressive palmar deviation of the third phalanx and subsolar abscesses in both forefeet. The gelding developed purulent discharge from the right coronary band and the hoof wall detached circumfrentially. Euthanasia was elected after 54 days. Continual exposure of the gelding's feet to ice water temperatures may have caused decreased perfusion and increased edema formation in the laminae resulting in decreased blood flow and exacerbating the existing ischemic necrosis.

Peripheral blood lymphocyte subpopulations and immunoglobulin concentrations in healthy foals and foals with Rhodococcus equi pneumonia.

Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses. Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young foals.

Pulmonary distribution of aerosolized technetium Tc 99m pentetate after administration of a single dose of aerosolized albuterol sulfate in horses with recurrent airway obstruction.

OBJECTIVE: To determine whether pulmonary distribution of aerosolized technetium Tc 99m pentetate is improved after inhalation of a single dose of albuterol sulfate in horses susceptible to recurrent airway obstruction (heaves). ANIMALS: 6 horses with heaves and 4 horses with normal respiratory tract function. PROCEDURE: Images were obtained during ventilation of horses at baseline (maximal change in pleural pressure during tidal breathing [deltaPpImax] >15 cm H2O) and after aerosolized albuterol sulfate (360 microg) administration, with a 24-hour washout period between experiments. The deltaPpImax was determined prior to the baseline scan, prior to albuterol sulfate administration, and 5 minutes after albuterol sulfate administration. Images were assessed by visual inspection (semi-quantitative scoring system) and histogram analysis. RESULTS: Images obtained from horses with heaves had nonuniform pulmonary distribution of radionuclide characterized by poor penetration in peripheral lung fields and excess deposition in large airways. Histogram analysis of images of the caudal portions of the lungs revealed nonuniform radionuclide deposition in horses with heaves and uniform radionuclide deposition in control horses. CONCLUSION: Administration of a single dose of aerosolized albuterol sulfate improved pulmonary distribution of aerosolized radiolabeled pentetate suspension in horses with heaves but did not alter pulmonary distribution in clinically normal horses. CLINICAL RELEVANCE: Precedent bronchodilator administration may improve pulmonary distribution of aerosolized, surface-active anti-inflammatory preparations.

Pulmonary function in horses with recurrent airway obstruction after aerosol and parenteral administration of beclomethasone dipropionate and dexamethasone, respectively.

OBJECTIVE: To determine changes in clinical signs of disease and response to pulmonary function testing in horses with recurrent airway obstruction (heaves) after aerosol and parenteral administration of beclomethasone dipropionate and dexamethasone, respectively. ANIMALS: 6 horses with inducible and reversible heaves. PROCEDURE: Episodes of heaves were induced by exposure (challenge) to moldy hay and straw for 7 days. Horses were assigned to treatment groups (aerosolized beclomethasone dipropionate, parenterally administered dexamethasone, aerosolized propellant [control]), and respiratory frequency and subjective assessment of respiratory effect were determined twice daily. Maximal change in pleural pressure (delta-Pplmax), pulmonary resistance (RL), and dynamic compliance (Cdyn) was determined on days 0, 7, 10, 14, and 21. RESULTS: The RL and delta Pplmax were increased, and Cdyn was decreased in all horses in response to natural challenge. Beclomethasone reduced RL on day 10, reduced delta Pplmax on days 14 and 21 and increased Cdyn on day 14. Dexamethasone reduced RL and delta Pplmax on days 10, 14, and 21 and increased Cdyn on days 10 and 14. Respiratory effort (subjective assessment) improved after 2 and 3 days of beclomethasone and dexamethasone administration but rebounded to pretreatment values 1 and 3 days after discontinuation of drugs. CONCLUSIONS: Pulmonary function testing responses and clinical signs of airway obstruction were improved by administration of beclomethasone. The magnitude of response to aerosolized beclomethasone generally was less marked than the response to parenterally administered dexamethasone. Higher or more frequent dosing of aerosolized beclomethasone may be necessary to achieve the anti-inflammatory response to parenterally administered dexamethasone.

Cytologic evaluation of bronchoalveolar lavage fluid from horses with recurrent airway obstruction after aerosol and parenteral administration of beclomethasone dipropionate and dexamethasone, respectively.

OBJECTIVE: To determine cytologic changes in horses with recurrent airway obstruction (heaves) after administration of aerosolized beclomethasone dipropionate and dexamethasone parenterally. ANIMALS: 6 horses with inducible and reversible heaves. PROCEDURE: Episodes of heaves were induced by exposure to moldy hay and straw for 7 days. Horses were assigned to treatment groups (aerosolized beclomethasone, parenterally administered dexamethasone, aerosolized propellant), and pulmonary inflammation was evaluated by serial cytologic examination of bronchoalveolar lavage (BAL) fluid samples obtained on days 0, 7, 10, 14, and 21. Total and differential cell counting and phenotypic analysis of lymphocyte subpopulations in BAL fluid were performed. RESULTS: 7 days of natural challenge induced neutrophilic inflammation. Neutrophil counts in BAL fluid were reduced in beclomethasone- and dexamethasone-treated horses on days 10 and 14 but rebounded to pretreatment values on day 21. The proportion of proinflammatory lymphocyte subpopulations (CD4+ and B+) and MHC class-II antigen expression were increased on days 14 and 21 in propellant-treated horses, compared with beclomethasone- and dexamethasone-treated horses. CONCLUSIONS: Aerosolized beclomethasone attenuated neutrophilic pulmonary inflammation and prevented alteration in lymphocyte subpopulations in horses with heaves. Results were similar to the response associated with parenterally administered dexamethasone. Short-term administration of aerosolized beclomethasone without minimizing environmental allergen exposure is not expected to provide prolonged anti-inflammatory benefit for horses with heaves.

Alteration in adrenocortical function in horses with recurrent airway obstruction after aerosol and parenteral administration of beclomethasone dipropionate and dexamethasone, respectively.

OBJECTIVE: To determine alteration in adrenocortical function in horses with recurrent airway obstruction (heaves) after aerosol and parenteral administration of beclomethasone dipropionate and dexamethasone, respectively. ANIMALS: 6 horses with inducible and reversible heaves. PROCEDURE: Episodes of heaves were induced by exposure to moldy hay and straw for 7 days (natural challenge). Horses then underwent treatment (aerosolized beclomethasone, parenterally administered dexamethasone, and aerosolized propellant) for 7 days. Horses remained in the mold-contaminated environment for 7 days after discontinuation of drugs. Adrenocortical function was determine by serial evaluation of cortisol concentration in serum obtained on days 0, 7, 9, 12, 14, 16, 19, and 21. Adrenocorticotropic hormone stimulation testing was performed in 4 horses/treatment group on days 0, 7, 14, and 21. RESULTS: Endogenous cortisol production was suppressed in beclomethasone- and dexamethasone-treated horses within 2 days of treatment but recovered to values similar to those in propellant-treated horses approximately 2 and 4 days after discontinuation of drugs. Serum cortisol concentration in propellant-treated horses gradually decreased during the study and was significantly lower than baseline on days 14, 16, 19, and 21. Mean increase in serum cortisol concentration in response to ACTH stimulation testing after beclomethasone and dexamethasone administration did not differ significantly from the response observed in propellant-treated horses. CONCLUSIONS: Aerosol and parenteral administration of beclomethasone and dexamethasone, respectively, suppressed adrenocortical function; however, endogenous cortisol production resumed approximately 2 and 4 days after discontinuation of drugs. Responsiveness to ACTH stimulation testing was not affected by the 7-day treatment period.

Immunologic function in horses after non-specific immunostimulant administration.

Inactivated Propionibacterium acnes is a biologic response modifier for treatment of non-specific respiratory disease in horses. The objectives of this investigation were to determine alterations in phagocytic activity, phenotypic expression of lymphocyte subpopulations and lymphokine-activated killing cell response in healthy young horses. Samples were collected on day 0, 7 and 14 of the investigation. Blood samples were obtained via jugular venipuncture and pulmonary leukocytes were recovered via bronchoalveolar lavage (BAL). Commercially available P. acnes (Eqstim) was administered intravenously on days 7, 9 and 11 of the investigation. Fever was observed on days 8 and 10, indicating immune reaction. Total peripheral blood white cell count was increased (P < 0.05) on day 14 after P. acnes administration compared to values on days 0 and 7. Total BAL fluid cell count decreased (P < 0.01) on day 14 compared to values on days 0 and 7, which was characterized by a decrease in total lymphocyte (P < 0.01) and macrophage (P < 0.01) counts. The proportion of lymphocytes in BAL fluid decreased (P < 0.005) on day 14 compared to values on days 0 and 7, and the proportion of macrophages increased (P < 0.005) on day 14 compared to values on days 0 and 7. P. acnes administration increased the total (P < 0.05) and proportional (P < 0.05) counts of CD4+ T lymphocytes in peripheral blood. Bronchoalveolar lavage fluid proportion of CD4+ (P < 0.05), CD5+ (P < 0.001) and MHC II (P < 0.05) lymphocytes increased on day 14 after P. acnes administration compared to values on days 0 and 7. Nonopsonized phagocytic activity in peripheral blood increased (P < 0.0005) on day 14 after P. acnes administration compared to values on days 0 and 7. Lymphokine-activated killing cell activity in peripheral blood and BAL fluid leukocytes was enhanced (P < 0.005) on day 14 after P. acnes administration compared to values on days 0 and 7. Serum IgG and IgM concentrations were within laboratory reference values and were not altered by administration of P. acnes. This investigation demonstrated immunostimulant and immunomodulatory properties of P. acnes, characterized by increased CD4+ T lymphocyte expression and LAK activity in peripheral blood and BAL fluid, increased nonopsonized phagocytosis in peripheral blood leukocytes and decreased pulmonary cellularity.

CD4+ and CD8+ T-lymphocytopenia in a filly with Pneumocystis carinii pneumonia.

Decreased proportion of CD4+ and CD8+ T lymphocytes in peripheral blood likely contributed to susceptibility to Pneumocystis carinii in a foal. Cytological evaluation of bronchoalveolar lavage was required for identification of the pathogen and serial flow-cytometric analysis of peripheral blood lymphocytes documented transient low expression of CD4+ and CD8+ T lymphocytes. Although immunodeficiency is uncommon, it must be included in the differential diagnosis for patients suffering from chronic or opportunistic infections and may provide an indication for immunostimulant therapy.

Fluid and electrolyte balance in endurance horses.

In horses, fluid losses of 10L to 15L per hour can occur during endurance exercise under hot environmental conditions. The horse's sweat is hypertonic with respect to plasma, and large amounts of sodium, chloride and potassium are lost during prolonged exercise. Horses that reach exhaustive syndrome often show pronounced increases in plasma protein concentration. This could be an indication of failure of the compensatory fluid shift from the intracellular compartment caused by severe dehydration. Thorough physical examination by the ride veterinarian allows early recognition of dehydration and electrolyte imbalances.

Developing an integrated information system for specialized addiction treatment agencies.

This article outlines the development of an integrated information system for specialized alcohol and drug treatment agencies in Ontario, Canada. The system is being developed following a strategic planning process involving provincial funding ministries and coalitions of service providers. An overview of the system's development is provided and the implementation of one subcomponent, a client-tracking system, is described. Some challenges to the implementation of this component are identified.

Systemic and colonic venous plasma biochemical alterations in horses during low-flow ischemia and reperfusion of the large colon.

The purpose of this study was to determine the effects of low-flow ischemia and reperfusion (I-R) of the large colon on 16 systemic venous (SV) and colonic venous (CV) plasma biochemical variables in horses. Horses (n = 24) were randomly allocated to 3 groups: sham-operated (n = 6), 6 h ischemia (n = 9), and 3 h ischemia followed by 3 h reperfusion (n = 9). SV and CV heparinized blood was collected at 0, 1, 3, 3.25, 4, and 6 h. The SV-CV difference was calculated for each variable. The SV, CV, and SV-CV difference for albumin, total protein, and calcium decreased significantly (P < 0.05) across time in horses of all groups, but there were no differences among groups. SV phosphorous was significantly increased from baseline (BL) at 1 to 6 h in horses of all groups, but there were no differences among groups. CV phosphorous was significantly greater than BL from 1 to 6 h in group-2 horses and from 1 to 3 h in group-3 horses. SV potassium was not different among groups, but was significantly higher at 6 h, compared with BL in horses of all groups. CV potassium was significantly greater than BL from 1 to 6 h in horses of groups 2 and 3. SV glucose was greater at 6 h compared with all previous times in horses of all groups, but there were no difference among groups. CV glucose was significantly lower than BL and group-1 values in horses of groups 2 and 3 during ischemia, but returned to BL during reperfusion in group-3 horses. CV anion gap was significantly greater and SV-CV anion gap was significantly more negative in horses of groups 2 and 3, compared with group-1 horses during ischemia. The biologic relevance of these alterations is unknown, but they may contribute to histopathologic, hemodynamic, and metabolic alterations characteristic of low-flow I-R. Alternatively, these alterations may simply reflect colonic injury sustained during I-R. Results suggest that the colon utilizes glucose as a fuel and generates acid anions during low-flow ischemia. Increased CV phosphorous and potassium during I-R likely occurs as a result of leakage of intracellular stores subsequent to cellular damage.