ABSTRACT: Reactive thrombocytosis is an increase in the circulating thrombocyte count secondary to a physiologic process within the body, often an infection. Reactive thrombocytosis is different than primary or essential thrombocytosis which is usually related to myeloproliferative neoplasia. Essential thrombocytosis is most common in adults, whereas reactive thrombocytosis is most frequently observed in children. Reactive thrombocytosis has been occasionally reported in cats, dogs and horses but has not been previously reported in the rabbit. Rabbits were challenged with virulent Pasteurella multocida. Hematologic, clinical, and culture assessments were performed prior to challenge, enabling each animal to serve as its own control. The questions asked were whether reactive thrombocytosis was a consistent phenomena and whether its presence and/or intensity was related to disease severity. All challenged rabbits demonstrated some degree of thrombocytosis in response to the infection, but individual rabbits were varied in their pattern of thrombocytosis. Elevations varied from intense to mild to undulating with durations of 1 to 11 days above 500x109/L and 0 to 5 days above 650x109/L. Correlation analysis was unable to demonstrate significant association between thrombocytosis, body temperature, leukocyte count, or the granulocyte lymphocyte ratio (all r < 0.2). No significant association between intensity of thrombocytosis and degree or type of pathologic lesions was observed. Thrombocytosis does not appear predictive of disease intensity or outcome. The data indicate that in the rabbit thrombocytosis is a consistent response to infection with P. multocida. Rabbits may serve as a model for the study of reactive thrombocytosis, in humans especially in children infected with Haemophilus sp., which are also a members of the bacterial family Pasteurellaceae.

Keywords: Animal model, Pasteurella multocida, platelets, rabbits, reactive thrombocytosis.

Reprint requests to: Dr. R.P. Ruble, Department of Population Health & Reproduction, School of Veterinary Medicine, University of California, Davis, CA, USA 95616, fax: (530) 752-5845, e-mail: rpruble@ucdavis.edu.

ABSTRACT: Several diseases have been related to oxidative stress. Recently, antioxidant functions have also been linked to anti-inflammatory properties. Cell defenses against reactive oxygen species include antioxidant enzymes. We studied the enzymatic antioxidant capacity in human blood of both red blood and mononuclear cells from patients suffering from an allergic reaction to pollen or house dust mite. We determined superoxide dismutases (SODs), glutathione peroxidase (GSHPx) and catalase (CAT) activities in each cell type. We also determined the extent of thiobarbituric acid reactive substances (TBARS), in order to study the correlation between the cellular enzymatic activities, the redox status and the disease. In mononuclear cells from allergic patients, SODs and CAT activities were enhanced compared to controls. Conversely, a decrease in GSHPx activity was found. In erythrocytes, higher values for GSHPx and SODs and similar CAT activities were found in allergic patients and controls. Interestingly, CuZnSOD and MnSOD activities were enhanced in the same proportion for both, erythrocytes and mononuclear cells. TBARS were also enhanced in both types of cells. The respective enzymatic imbalances in mononuclear cells and erythrocytes, namely, GSHPx/SOD and CAT/SOD, and their consequences are discussed. To our knowledge, this is the first global study of antioxidant enzymes, including TBARS level determinations, in allergy.

Keywords: Allergy, catalase, CuZn superoxide dismutase, glutathione peroxidase, house dust mite, human blood cells, lipid peroxidation, Mn superoxide dismutase, pollen, reactive oxygen species.

Reprint requests to: José M. Matés, Department of Molecular Biology and Biochemistry, Sciences Faculty, University of Málaga, Campus de Teatinos, s/n 29071 Málaga, SPAIN, phone: +34-95-213-7135, fax: +34-95-213-2000, e-mail: jmates@uma.es.

ABSTRACT: Our current strategy for gene therapy of sickle cell anemia involves retroviral vectors capable of transducing "designer" globin genes that code for novel anti-sickling globins (while resisting digestion by a ribozyme), coupled with the expression of a hammerhead ribozyme that can selectively cleave the human betaS mRNA. In this report, we have tested in vivo an anti-betaS hammerhead ribozyme embedded within a cDNA coding for the luciferase reporter gene driven by the human Beta-globin promoter and hyper-sensitive sites 3 and 4 of the locus control region. We have created mice transgenic for this luciferase-ribozyme construct and bred the ribozyme transgene into mice that were already transgenic for the human betaS gene. We then measured expression of the betaS transgene at the protein and RNA levels by HPLC and primer extension. The presence of the ribozyme was associated with a statistically significant reduction in the level of betaS mRNA in spleen stress reticulocytes (from 60.54.1% to 52.9 4.2%) and in the percentage of betaS globin chains in very young mice (from 44.5 +/- 0.6% to 40.8 +/- 0.7%). These results demonstrate that it is possible to decrease the concentration of betaS chains and mRNA with the help of a hammerhead ribozyme. While the enormous amount of globin mRNA in reticulocytes is a challenge for ribozyme technology, the exquisite dependence of the delay time for formation of Hb S nuclei on the concentration of Hb S in red blood cells suggests that even a modest reduction in Hb S concentration would have therapeutic value.

Keywords: Hammerhead ribozyme, sickle cell anemia, beta-globin, gene therapy, mouse model.

Reprint requests to: Eric Bouhassira, Ph.D., Division of Hematology, U921, Albert Einstein College of Medicine, 1300 Morris Park Ave. Bronx, New York 10461, phone: (718) 430-2188, fax: (718) 824-3153, email e-mail: bouhassi@aecom.yu.edu.