The use of cytochemistry,immunophenotyping, flow cytometry,and in vitro differentiation to determine the ontogeny of a canine monoblastic leukemia

We evaluated the utility of cytochemistry, immunophenotyping, flow cytometry, and in vitro culture with forced differentiation of leukemic cells as diagnostic aids to identify the malignant cell ontogeny in a dog with leukemia. A tentative diagnosis of monoblastic leukemia was established by microscopic examination of Romanowsky-stained blood smears and bone marrow aspirate smears. This diagnosis also was supported by the light scatter signature that identified the blast cells as large, non-granular monocytic cells using a CellDyn 3500 automated hematology analyzer; as well as by the detection of N-butyrate esterase and the lack of choloroacetate esterase or leukocyte peroxidase by cytochemical staining. Subsequently, leukemic cells were isolated from the dog's peripheral blood and placed into tissue culture or cryopreserved. The leukemic cells grew in suspension cultures and proliferated spontaneously for up to 4 days. By day 7, proliferation was negligible. Upon culture with conditioned supernatant using mitogen-stimulated human T cells as a source of cytokines, an increased proportion of cells entered S phase by day 2 of culture; however, proliferation declined markedly by day 4, at which time the cells had apparently differentiated to adherent, vacuolated macrophages. The cytokine-stimulated leukemic cells were positive for the monocyte/macrophage specific markers alpha-1-antitrypsin, alpha-1-antichymotrypsin, lysozyme, CD14, MHC class II, and calprotectin, an antigen found in differentiated macrophages and granulocytes. Despite the strong tendency of the leukemic cells towards monocytic differentiation, our results suggested that they retained some features of a myelomonocytic precursor. These data show that cytochemistry, immunophenotyping, flow cytometry, and in vitro differentiation of canine leukemia cells are useful tools for confirming the lineage of malignant hematopoietic cells.     

Control and management of sheep mange and pediculosis in Great Britain

The patterns of insecticide use for the prevention and control of sheep lice and Psoroptes mites, in England, Wales and Scotland, in 2003-2004, were examined using data from a retrospective questionnaire. Overall, 375 of the 966 (39%) farmers who responded said that they treated at least once to prevent louse infestation. For scab, 426 of the 966 (44%) farmers who responded said that they had treated prophylactically to prevent the disease. There were no significant differences between regions in the number of times farmers treated to prevent lice (chi(2)=0.54, P>0.1) or scab (chi(2)=0.16, P>0.1). Among the farmers who applied prophylactic treatment 23% had a subsequent outbreak of lice and 17% had an outbreak of scab. The majority of sheep farmers who used preventative treatment did so in June and July. Where a second treatment was given it was largely in October. For lice, 52% of farmers used pyrethroids to prevent and 70% to treat louse infestation. For scab prophylaxis, most farmers said that they used either the macrocyclic lactones (41%) or organophosphates (36%) as a preventative. For the therapeutic treatment of scab outbreaks, macrocyclic lactones (largely doramectin) were used on 58% of sheep farms. The high national prevalence of sheep scab and pediculosis indicates that current control methods are failing to contain these ectoparasites. The results of this study suggest that a large proportion of insecticide applications take place at times of year when they might expect to be relatively ineffective, and the use of inappropriate compounds for the parasites in question is common. The widespread use of macrocyclic lactones in particular for scab prevention and control is of concern, because of the additional use of these compounds in parasitic worm control and the potential that their overuse will hasten the development of resistance.     

Surgical repair of nasopharyngeal stenosis in a cat using a stent.

An intraluminal stent was used to maintain patency of a recurrent nasopharyngeal stenosis (NPS) in a cat. The stenotic membrane within the nasopharynx was resected, and a 2-cm long, braided-wire endoprosthesis was placed as a stent. The patient was evaluated at one day, six weeks, 19 weeks, and 49 weeks following surgery. The cat tolerated the stent well. The 19-week recheck revealed granulation tissue partially obstructing the pharyngeal aspect of the stent which was subsequently surgically resected. Complications after excision of the granulation tissue included intermittent upper respiratory congestion and nasal discharge. The 49-week recheck showed no increased granulation tissue; however, upper respiratory congestion was still present. This particular stent, and its use as described in this paper, is recommended in cases of chronic recurrent NPS.     

Evaluation of the estrogenic effects of legume extracts containing phytoestrogens

Seven legume extracts containing phytoestrogens were analyzed for estrogenic activity. Methanol extracts were prepared from soybean (Glycine max L.), green bean (Phaseolus vulgaris L.), alfalfa sprout (Medicago sativa L.), mung bean sprout (Vigna radiata L.), kudzu root (Pueraria lobata L.), and red clover blossom and red clover sprout (Trifolium pratense L.). Extracts of kudzu root and red clover blossom showed significant competitive binding to estrogen receptor beta (ERbeta). Estrogenic activity was determined using an estrogen-dependent MCF-7 breast cancer cell proliferation assay. Kudzu root, red clover blossom and sprout, mung bean sprout, and alfalfa sprout extracts displayed increased cell proliferation above levels observed with estradiol. The pure estrogen antagonist, ICI 182,780, suppressed cell proliferation induced by the extracts, suggesting an ER-related signaling pathway was involved. The ER subtype-selective activities of legume extracts were examined using transiently transfected human embryonic kidney (HEK 293) cells. All seven of the extracts exhibited preferential agonist activity toward ERbeta. Using HPLC to collect fractions and MCF-7 cell proliferation, the active components in kudzu root extract were determined to be the isoflavones puerarin, daidzin, genistin, daidzein, and genistein. These results show that several legumes are a source of phytoestrogens with high levels of estrogenic activity.     

Improving bioavailability and stability of genistein by complexation with high-amylose corn starch

Genistein, like other phytochemicals, has beneficial health effects, but its bioavailability is limited. This research studied the effect of complexation of genistein with starch on genistein bioavailability. Genistein release from these complexes was tested in vitro under simulated intestinal conditions and in vivo in rats fed high-amylose corn starch (HACS)-genistein complexes (experimental group) as compared to those fed a physical mixture of HACS and genistein (controls). In vitro results showed that genistein release is sustained and fits the normal transit time of food in the intestine. The genistein concentration in the plasma was twice as high in the experimental group versus controls; the genistein concentration in the urine was also higher in the experimental group but lower in the feces. These results indicate that starch-genistein complexes increase genistein bioavailability and suggest that starch can affect the bioavailability of additional food components.