Evaluation of cardiopulmonary and inflammatory markers in dogs with heartworm infection treated using the slow kill method.

This study evaluated the changes in the levels of cardiac, hemostatic, and inflammatory biomarkers in 12 dogs with different severities of heartworm infection treated using the slow kill protocol, consisting of 6-10µg/kg of ivermectin and 10mg/kg of doxycycline combination. The serum levels of cardiac troponin-I, D-dimer, C-reactive protein, and interleukin-6 were measured on the day of diagnosis (D0), after termination of doxycycline administration (D30), after termination of the slow kill treatment (D180), and 10 months after the initiation of therapy (D300). Heartworm antigenemia was cleared in 4/4 class I dogs, 3/4 class II dogs, and 1/4 class III dogs at the end of the therapy (D180), and in 4/4 class I, 4/4 class II, and 1/4 class III dogs at the end of the study (D300). The serum levels of the markers in class I dogs on the day of diagnosis (D0) were within the reference range, while the levels in class II and III dogs were above the reference range. Further, the serum levels of the markers in all dogs decreased significantly at the end of the study (D300), although some markers in class III dogs remained at pathological levels. This study revealed that the slow kill method should be used only as an alternative therapeutic protocol for dogs with low worm burden (class I and II). As the slow kill method alone may not effectively reduce all pathological changes in dogs with heavy worm burden and severe clinical signs (class III), adjuvant therapies including steroids and anti-thromboembolics should be used to minimize the risk of complications.

Comparison of 2 retrieval devices for heartworm removal in 52 dogs with heavy worm burden.

BACKGROUND: For treating dogs with heavy heartworm infection, mechanical removal using various retrieval devices is useful. However, the efficacy and safety of retrieval devices have rarely been studied. HYPOTHESIS: Catheter-based heartworm removal using 2 retrieval devices (basket and tripod grasping forceps) is efficient and safe for treating dogs with heavy worm burden. ANIMALS: Fifty-two client-owned dogs with heavy (Class III and IV) worm burden. METHODS: A retrospective study was performed on 52 dogs, using a catheter-based heartworm removal approach using 2 types of retrieval devices (ie, the basket and the tripod grasping forceps). The efficacy and complications associated with the 2 devices were assessed. RESULTS: The basket device was used on 22 of the study group dogs, and the tripod grasping forceps was used on 30 of the dogs. The postoperative survival rate was 95.5% for the basket device and 80% for the tripod grasping forceps, but the difference was not statistically significant. The worm number captured per attempt was 3.5 ± 1.7 using the basket device and 1.9 ± 0.85 for the tripod grasping forceps (P < .05). Various complications associated with heartworm removal were noticed with both retrieval devices. CONCLUSIONS AND CLINICAL IMPORTANCE: This study suggests that catheter-based heartworm removal is not only a relatively safe and efficient therapeutic method in dogs with heavy worm burden, but more efficient using the basket device. Our data do not indicate a clear safety advantage between the 2 devices evaluated, although the survival rate was numerically higher in dogs undergoing a basket intervention.

Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus.

We investigated the mechanism of the immunomodulatory action of polysaccharide (ASP) isolated from a cell culture of Acanthopanax senticosus. ASP was found to directly increase the proliferation and differentiation of B cells, and the cytokine production of macrophage, but not the proliferation and cytokine production of T cells. Since ASP cannot penetrate the cell membrane due to its large molecular mass, such cellular activation may be caused by the surface binding of ASP to receptors expressed on B cells and macrophages. The possibility that TLRs, which are known to be involved in immune-related responses, may be the receptor(s) of ASP was investigated. The immunomodulating activities of ASP on the B cells and macrophages of C3H/HeJ mice, expressing a defective toll-like receptor (TLR)-4, were decreased versus the corresponding cells from C3H/HeN mice. In addition, the activities of ASP on B cells and macrophages were significantly reduced by treating the cells with antibodies to TLR4 and TLR2 prior to ASP, suggesting that both of them are the possible receptors of ASP. The ligation of TLRs induced by ASP was able to activate mitogen-activated protein kinases (MAPKs), such as Erk1/2, p38 and JNK, and the transcription factor NF-kappaB. Although ASP was shown to activate the TLR signaling cascades in the same manner as lipopolysaccharide (LPS), these two could be differentiated by the finding that polymyxin B (PMB), a specific inhibitor of LPS, did not significantly affect the activities of ASP on B cells and macrophages. Taken together, our results demonstrate that ASP, isolated from a cell culture of A. senticosus, activates B cells and macrophages by interacting with TLRs and leading to the subsequent activation of mitogen-activated protein kinases and NF-kappaB.

Modulation of transferrin synthesis, transferrin receptor expression, iNOS expression and NO production in mouse macrophages by cytokines, either alone or in combination.

Iron, an essential element for all living organisms, is central importance in a number of crucial metabolic pathways, including the regulation of immune function. Iron delivery to cells is accomplished by the complexing of iron to transferrin (Tf), a monomeric iron-binding protein in the plasma, followed by specific binding of Tf to cell-surface receptors, endocytosis of the receptor-ligand complexes and ultimately, release of iron from endosomal vesicles to the cytoplasm. The purpose of this study was to evaluate the effect of cytokines, alone and in combination, on the factors that can affect the iron delivery in thioglycollate-elicited macrophages. In this study, IFN gamma induced a marked increase in Tf synthesis by macrophages, while IL-1, IL-6 and TNF alpha produced a more modest increase. Combinations of these cytokines were shown to be less effective in promoting macrophage Tf synthesis than the cytokines by themselves. IFN gamma alone and in combination with other cytokines was effective in inducing nitrite (NO) production and inducible nitric oxide synthetase (iNOS) expression in macrophages, while IL-1, TNF alpha and IL-6 individually, as well as in various combinations, were not. While all tested cytokines individually and in combination inhibited the expression of the transferrin receptor (TfR) on macrophages, IFN gamma alone and in combination with other cytokines most strongly repressed the TfR expression. TfR localization in macrophages after IFN gamma stimulation showed that TfR fluorescence was most intense in the perinuclear region after 6 hours and scattered diffusely throughout the cytoplasm after 24 hours. This data suggests that IFN gamma may enhance iron uptake during the early phase of macrophage activation, and in later phases, down-regulate TfR expression by inducing NO, thus contributing to intracellular oxidative stress reduction.

Somatostatin and substance P induced in vivo by lipopolysaccharide and in peritoneal macrophages stimulated with lipopolysaccharide or interferon-gamma have differential effects on murine cytokine production.

We have investigated whether lipopolysaccharide (LPS) induces substance P (SP) and somatostatin (SOM) in popliteal lymph nodes in vivo and whether macrophages are a source of SP and SOM in vitro. We have also investigated the effect of SP and SOM treatment on the production of cytokines. SP reached a maximum 3 days after injection of LPS (100 microg/footpad) and then declined. SOM expression after LPS injection reached a maximum at 5-7 days. Stimulation of thioglycolate-elicited peritoneal macrophages with LPS (20 microg/ml), recombinant interferon-gamma (rIFN-gamma, 100 U/ml), and LPS plus rIFN-gamma induced SOM and SP. Thioglycolate-elicited, unstimulated peritoneal macrophages also synthesized these peptides. SOM (10(-12)-10(-8) M) significantly inhibited IL-6 and IFN-gamma production, whereas SP at those concentrations enhanced cytokine production by activated lymphocytes and macrophages. These findings suggest that neuropeptides which originate from macrophages and nerve fibers act as immunomodulators to mediate changes in the pattern of cytokine production.

Effects of transferrin on the modulation of cytokine production on mouse spleen cells.

In order to investigate the the effects of transferrin(Tf) on the production of cytokines, mouse spleen cells were treated with various concentrations of apo- and holo-Tf, and then the production of IL-6, IFN gamma and the expression of mRNA for TNF alpha was determined. The distribution of Tf, macrophages and T cells in the mouse mammary glands was also examined. IL-6 and IFN gamma producing capabilities of the unstimulated spleen cells in the presence of apo and holo-Tf were increased in a dose dependent manner, while the cells stimulated with anti-CD3 had no significant effects on production in thd presence of graded concentrations of Tf. The relative abundance of TNF alpha mRNA was significantly affected by the concentration of TF. During early involution almost all of the secretory epithelial cells and the secretion in the alveoli showed a very strong positive reaction to transferrin antibody, and macrophages and T cells were distributed in the lumen, alveolar epithelial layer and connective tissue area. These findings suggest that the upregulated patterns of these cytokines and distribution of immune cells may play a beneficial role in the augmentation of host's defense mechanisms during involution.

307-bp fragment in HOXA7 upstream sequence is sufficient for anterior boundary formation.

The HOX genes are expressed in a positionally and temporally restricted manner involving anteroposterior axial pattern formation during early embryogenesis. Previously, we studied the sequence and function of an upstream regulatory region of the human HOXA7 gene. To identify a critical cis-acting element, a deletion analysis was performed along the human control region (HCR) (about 1.1 kb), which was sufficient for setting the anterior boundary of expression in transgenic mice. We demonstrated that a 307-bp control region contains a cis-acting element(s) specifying an anterior boundary as well as a dorsal-ventral restriction in the neural tube at day 12.5 postconception (p.c.). The distinct anterior limit of expression was noted at the level of C7/T1 in the neural tube and spinal ganglia. In addition, our deletion experiments revealed that the HCR consisted of several cis-acting elements which were individually capable of driving regionally restricted expression patterns in the neural tube and limb buds.