Macrophage effector mechanisms mediating acute graft-versus-host disease

The study presented in this thesis is an investigation of M$phi$ effector mechanisms that mediate the pathogenesis of acute graft-versus-host disease (GVHD). Since the time that the GVH reaction was first described the mechanisms underlying the development of the disease have not been characterized. We have examined the state of M$phi$ activation in nonirradiated B6AF1 mice injected with either 60 $times$ 10$sp6$ (acute GVHD) or 30 $times$ 10$sp6$ (nonlethal GVHD) parental B6 lymphoid cells. TNF-$alpha$ and nitric oxide (NO) generation by M$phi$ occurs following priming by IFN-$gamma$ and triggering by LPS. During the early phase of acute GVHD, administration of normally sublethal amounts of LPS triggered release of significant amounts of TNF-$alpha$ into the serum resulting in death of the animals within 36 hours. The amount of serum TNF-$alpha$ produced following LPS injection increased during the course of GVHD and was significantly greater in acute GVH reactive mice. Normal animals treated with the same dose of LPS neither died nor produced detectable amounts of serum TNF-$alpha$. In vitro studies demonstrated that M$phi$ undergo priming for both TNF-$alpha$ and NO production during the developing GVH reaction and that priming was greater during acute GVHD than nonlethal GVHD. As a result of M$phi$ priming during acute GVHD, M$phi$ mediated the selective release of iron from $sp{59}$Fe,$sp{51}$Cr dual-labelled target cells and expressed cytostatic activity when triggered by LPS. NO generation and cytostasis mediated by M$phi$ from acute GVH-reactive animals were inhibited by NMMA. Endogenous bacterial-derived LPS was detected in the livers and spleens of acute GVH reactive animals and appeared subsequently the serum coincident with the onset of mortality. Immunohistochemical staining for TNF-$alpha$ during acute GVHD demonstrated TNF-$alpha$ in the splenic red pulp and liver, however, the hepatic portal infiltrates that characterize acuteOur results demonstrate the entry of bacterial-derived LPS during the acute GVH reaction and that as a result of M$phi$ priming, LPS in the transplant recipient acts as a trigger for TNF-$alpha$ and NO production by M$phi$. The presence of enteric Gram-negative bacteria is a risk factor for development of acute GVHD due to the triggering effect of LPS on M$phi$ that are primed following bone marrow transplantation. The development of acute GVHD therefore involves the excessive priming of M$phi$ followed by contact with LPS leading to the activation of NO and TNF-$alpha$-mediated mechanisms of tissue injury, weight loss, septic shock and death of the transplant recipient

Platelet Toll-like receptor expression modulates lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-α production in vivo

Toll-like receptors (TLRs) play a critical role in stimulating innate immunity by recognizing pathogen-associated molecular patterns (PAMPs) on invading microorganisms. Platelets also play a role in innate immunity, and we studied whether they express TLR. Results show that human and murine platelets variably expressed TLR2, TLR4, and TLR9 by flow cytometry and Western blotting. TLR4 expression was confirmed by demonstrating murine platelet binding to lipopolysaccharide (LPS). Thrombin activation of the platelets significantly enhanced the expression of TLR9, suggesting that at least some TLRs may derive from intracellular compartments. When LPS was administered to LPS-sensitive C3H/HeN and LPS-resistant C3H/HeJ mice, functional TLR4 expression in vivo was shown to be responsible for LPS-induced thrombocytopenia. However, when the C3H/HeN mice were first rendered thrombocytopenic by an antiplatelet antibody and then administered LPS, a significant reduction occurred in their ability to produce TNF-α. The decreased cytokine production in the thrombocytopenic mice was restored with platelet transfusion. These results suggest that platelets express various TLRs and that the functional significance of one of these, TLR4, appears to be a role in the modulation of LPS-induced thrombocytopenia and TNF-α production. This work implicates platelets as important mediators of innate immune responses against invading microorganisms

Pharmacokinetics and bioavailability of the isoflavone biochanin A in rats

Biochanin A(BCA) is a dietary isoflavone present in legumes, most notably red clover, and in many herbal dietary supplements. BCA has been reported to have chemopreventive properties and is metabolized to the isoflavone genistein (GEN), BCA conjugates, and GEN conjugates. The metabolites may contribute to the chemopreventive effects of BCA. The absorption, metabolism, and disposition of BCA have not been determined in rats. Our objective was to evaluate the pharmacokinetics and metabolism of BCA in rats. Male Sprague-Dawley rats were administered BCA by intravenous injection (1 and 5 mg/kg), by intraperitoneal injection (5 and 50 mg/kg), and orally (5 and 50 mg/kg). Plasma and bile samples were enzymatically hydrolyzed in vitro to determine conjugate concentrations for BCA and GEN. Equilibrium dialysis was used to determine protein binding. The BCA and GEN concentrations in plasma, urine, and bile were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The pharmacokinetic parameters of BCA were analyzed by noncompartmental analysis. Significant levels of BCA conjugates and GEN conjugates were detected in plasma and bile. Both BCA and GEN were found to have a high clearance and a large apparent volume of distribution; the bioavailability of both was poor (<4%). Reentry peaks were evident after oral administration of both BCA and GEN, suggesting enterohepatic cycling. The free fraction of BCA in rat plasma was 1.5%. A2-compartment model that included both linear and nonlinear clearance terms and enterohepatic recirculation best described the plasma data. This represents the first evaluation of the dose-dependent pharmacokinetics and metabolism of BCA in rats