Decreased thymopoietic capacity contributes to the severe and clinically significant immune deficiency seen after bone marrow transplantation (BMT). One mechanism for thymopoietic failure is damage to the interleukin 7 (IL-7)-producing thymic epithelial cells (TECs) by irradiation and chemotherapy, which can be partially treated by IL-7 administration. Pretreatment of BMT recipients with kera-tinocyte growth factor (KGF, or Fgf7), an epithelial cell-specific growth factor, protects mucosal, cutaneous, and pulmonary epithelial cells from cytotoxic therapy-induced damage in experimental murine models. Like other epithelial cells, TECs specifically express KGF receptors. Because KGF specifically protects KGF receptor-bearing epithelial cells and post-BMT immune deficiency is caused by loss of TECs, we hypothesized that KGF pretreatment would improve post-BMT thymic function. To test the hypothesis, BMT recipient mice were given KGF or placebo prior to congenic or allogeneic BMT. Administration of KGF before mu-rine BMT significantly increased the capacity of the thymus to generate donor-derived thymocytes. KGF pretreatment also normalized the proportion of thymic subpopulations, increased the number of naive T cells in the periphery, and improved the response to neoantigen immunization. KGF treatment caused increased production of intrathymic IL-7, and the thymopoietic effects of KGF required an intact IL-7 signaling pathway. These results demonstrate that KGF may have immunomodulatory effects by a unique mechanism of protection of TECs. Furthermore , thymic injury and prolonged posttransplantation immune deficiency in BMT recipients can be prevented by KGF administration. (Blood. 2002;99:4592-4600)

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It is generally assumed that there is symmetric distribution of the glucose transporter on the lumenal and ablumenal membranes of the brain capillary endothelial cell that makes up the blood-brain barrier (BBB) in vivo. However, the presence of brain endothelial tight junctions allows for asymmetric distribution of BBB plasma membrane proteins. Glucose transporter isoform 1 (GLUT-1), the prinicipal glucose transporter at the BBB, was assessed in rat brain in the present studies using immunogold electron microscopy. The distribution of the immunoreactive GLUT-1 protein on the endothelial lumenal membrane, the ablumenal membrane, and the cytoplasmic compartment was 12%, 48%, and 40%, respectively, and no significant immunolabeling of the neuropil was measurable. These studies suggest (i) that GLUT-1 is asymmetrically distributed on the BBB plasma membrane with an 4-fold greater abundance on the ablumenal membrane as compared to the lumenal membrane; (ii) that "40% of the endothelial glucose transporter protein is contained within the cytoplasmic space, which provides a mechanism for rapid up-regulation of the transporter by altered distribution of transporter between cytoplasmic and plasma membrane compartments; and (iii) that no significant labeling of neuropil is found with antisera directed against the GLUT-1 protein.These studies also suggest mechanisms of regulation of glucose transport from blood to brain that involve differential distribution of the BBB glucose transporter in subcellular compartments of brain capillary endothelial cells.The major metabolic substrate of the brain is glucose, and the transport of glucose from blood to brain is tightly coupled with cerebral glucose utilization (1). The transport of this important molecule into brain tissue is regulated at the brain capillary endothelial wall-i.e., the blood-brain barrier (BBB) (2, 3). Glucose transport through this barrier is accomplished by the facilitated diffusion through two membranes in series, the lumenal and ablumenal endothelial membranes (4), which are separated by -0.3 ,um of cytoplasm. Recent studies have established that glucose transporter isoform 1 (GLUT-1), which is a member of a sodiumindependent glucose transporter gene superfamily (5), is quantitatively the most important, if not the only, glucose transporter isoform on the brain capillary endothelium (6-8).Pappenheimer and Setchell (4) emphasized that the transport of glucose through the BBB, although operationally treated as transport through a single membrane, actually represents transport through a double-membrane barrier. The single-membrane treatment has been used in a number of physiologic models of BBB glucose transport because (i) glucose equilibrates with the entire endothelial volume within milliseconds (9), since this volume is only -0.1% of the total brain volume (10), and (ii) it is generally found that the kinetic parameters ofglucose transport from blood-to-brain and from brain-to-blood are identical (4, 9, 11, 12). However, the maximal transport capac...

Thymus-dependent reconstitution of the peripheral T-cell compartment is critical for the successful outcome of bone marrow transplantation. However, graft-versus-host disease (GVHD) affects thymic stromal function and thus prevents normal T-cell maturation and selection. To determine whether cytoprotection of thymic epithelial cells (TECs) by keratinocyte growth factor (KGF) averts GVHD-related injury to the thymus, a nonirradiated murine parent-->F(1) transplantation model was investigated. Administration of KGF between days -3 and +3 of GVHD induction preserved normal thymic size, cellularity, and thymocyte phenotype when measured 2 weeks after transplantation and compared with saline-treated parent-->F(1) mice that received allogeneic transplants. Moreover, the characteristic GVHD-induced impairment in cell cycle progression of pro- and pre-T cells was prevented by KGF. However, the normal phenotypic and functional status of the thymus did not correlate with the higher number of GVHD-inducing mature donor T cells in thymi of KGF-treated mice. Importantly, extensive analysis of the different TEC populations within the thymic cortex and medulla revealed an almost normal stromal architecture and composition in GVHD mice treated with KGF. These observations are likely to reflect an indirect effect of KGF on thymopoiesis as KGF-receptor expression was demonstrated to be restricted to TECs. Thus, pharmacologic doses of KGF appear to exert a potent effect on TEC function, which in turn allows for normal T lymphopoiesis to occur during acute GVHD.

Banks, William A., and Catherine L. Farrell. Impaired transport of leptin across the blood-brain barrier in obesity is acquired and reversible. Am J Physiol Endocrinol Metab 285: E10-E15, 2003. First published March 4, 2003 10.1152/ ajpendo.00468.2002.-Leptin resistance is a major cause of obesity in humans. A major component of this resistance is likely an impaired transport of leptin across the blood-brain barrier (BBB). The fattest subgroup of otherwise normal 12-mo-old CD-1 mice have severely impaired transport of leptin across the BBB. However, it is unknown whether these mice are born with a BBB impairment or acquire it with aging and obesity. Here, we found within an otherwise normal population of CD-1 mice that the 10% fattest mice gained weight throughout a 12-mo-life span, whereas the 10% thinnest mice gained little weight after 3 mo of age. The fattest mice acquired a progressive impairment in their ability to transport leptin across the BBB, whereas the thinnest mice had a rate of transport that did not change with age. Fasting fat mice for 24 h or treating them with leptin resulted in modest weight reduction and development of transport rates for leptin across the BBB similar to those of thin mice. These results show that, in obese CD-1 mice, the impaired transport of leptin across the BBB develops in tandem with obesity and is reversible with even modest weight reduction. resistance syndrome; peptide; anorexia; fasting HUMAN OBESITY represents a resistance state to leptin. Resistance is likely caused by a combination of resistance at the receptor and postreceptor levels as well as a decreased ability of the blood-brain barrier (BBB) to transport circulating leptin into the brain. Several studies have documented various types of evidence for impaired transport of leptin across the BBB. For example, obese humans have a decreased cerebrospinal fluid-to-serum ratio for leptin (8,22), and some obese rats who no longer respond to peripherally administered leptin can still respond to leptin given directly into the central nervous system (11, 23). Direct impairment of leptin transport across the BBB has been shown in obese rats and mice (5,7,9,17).The question remains whether defects in leptin transport are inherent in animals destined to become obese or are acquired with the obesity. This question is key to understanding the role of the BBB in leptin resistance and the treatment of obesity. If the leptin transporter is defective before the onset of obesity, then this would suggest that the defect plays a fundamental role in the development of obesity. It would further suggest that the propensity to obesity and the degree to which an individual will become obese can be determined by examination of BBB properties. If the BBB defect is acquired, then this would suggest that some factor, perhaps reversible, that arises with the development of obesity induces the BBB defect. A positive feed-forward cycle of increasing obesity, BBB transport impairment, and leptin resistance could arise. Such an acquired impai...

The present studies describe the biodistribution of cationic liposomes and cationic liposome/oligonucleotide complex following intravenous injection into mice via the tail vein. (111)In-diethylenetriaminepentaacetic acid stearylamide ((111)In-DTPA-SA) was used as a lipid-phase radiolabel. Inclusion of up to 5 mol% DTPA-SA in liposomes composed of 3beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) did not influence liposome formation or size, nor the binding/uptake or fusion of the cationic liposomes with CHO cells in vitro. Moreover, nuclear delivery of oligonucleotide to CHO cells was unaffected by the probe. The biodistribution of liposomes with increasing concentration of DC-Chol (1:4-4:1, DC-Chol/DOPE, mol/mol) at 24 h post-injection revealed no dependence on lipid composition. Uptake was primarily by liver, and accumulation in spleen and skin was also observed. Comparatively little accumulation occurred in lung. Clearance of injected liposomes by liver was very rapid (approximately 84.5% of the injected dose by 7.5 h post-injection). Liposome uptake by liver and spleen were equally efficient in the dose range of 3.33 to 33.33 mg/kg body weight, yet possible saturation of liver uptake at a dose of 66.80 mg/kg may have allowed for increased spleen accumulation. Preincubation of cationic liposomes with phosphorothioate oligonucleotide induced a dramatic yet transient accumulation of the lipid in lung which gradually redistributed to liver. Similar results were observed when monitoring iodinated oligonucleotide in the complex. Immuno-histochemical studies revealed large aggregates of oligonucleotide within pulmonary capillaries at 15 min post-injection, suggesting the early accumulation in lung was due to embolism. Immuno-histochemical studies further revealed labeled oligonucleotide to be localized primarily to Kupffer cells at 24 h post-injection. Immuno-electron microscopy revealed localization of oligonucleotide primarily to the lumen of pulmonary capillaries at 15 min post-injection. Immuno-electron microscopy revealed localization of oligonucleotide primarily to the lumen of pulmonary capillaries at 15 min post-injection, and to phagocytic vacuoles of Kupffer cells at 24 h post-injection. By these methods, nuclear delivery of oligonucleotide in vivo was not observed. Increasing concentration of mouse serum inhibited cellular binding/uptake of cationic liposomes in vitro, without or with complexed oligonucleotide. We therefore postulate that interaction with plasma components, including opsonin(s), inhibits cellular uptake of the injected liposomes as well as the liposome/oligonucleotide complex, and mediates rapid uptake by Kupffer cells of the liver. These results are relevant to the design of cationic liposomes for efficient delivery of nucleic acid in vivo.

Impaired blood-brain barrier transport of leptin into the arcuate nucleus has been suggested to underlie obesity in humans and outbred aging mice. Here, we used a brain perfusion method in mice to measure transport rates and kinetic parameters for leptin at vascular concentrations between 0.15 and 130 ng/ml. Transport into whole brain was partially saturated at all concentrations, not only those seen in obesity. Leptin entered all regions of the brain, not only the hypothalamus, with entry and saturation rates differing among the brain regions. The value of the Michaelis-Menten constant of the transporter approximates normal serum levels and the maximum velocity value varies significantly among brain regions. These results suggest an important role for low serum levels signaling starvation status to the brain and show that the levels of leptin seen in obesity greatly saturate the transporter. Differences in regional uptake and saturation provide a mechanism by which leptin can control events mediated at the arcuate nucleus and other regions of the central nervous system with different regional thresholds for optimal function.

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