The involvement of the blood-brain and the blood-cerebrospinal fluid barriers in the distribution of leptin into and out of the rat brain.

Leptin is a 16 kDa hormone that is produced by adipose tissue and has a central effect on food intake and energy homeostasis. The ability of leptin to cross the blood-brain and blood-cerebrospinal fluid (CSF) barriers and reach or leave the CNS was studied by the bilateral in situ brain perfusion and isolated incubated choroid plexus techniques in the rat. Brain perfusion results indicated that [(125)I]leptin reached the CNS at higher concentrations than the vascular marker, confirming that [(125)I]leptin crossed the brain barriers. Leptin distribution varied between CNS regions and indicated that the blood-brain barrier, in contrast to the blood-CSF route, was the key pathway for [(125)I]leptin to reach the brain. Further perfusion studies revealed that [(125)I]leptin movement into the arcuate nucleus, thalamus, frontal cortex, choroid plexuses and CSF was unaffected by unlabelled human or murine leptin at a concentration that reflects the upper human and rat plasma leptin concentration (2.5 nM). In contrast, the cerebellum uptake of [(125)I]leptin was decreased by 73% with 2.5 nM human leptin. Thus, this site of dense leptin receptor expression would be sensitive to physiological changes in leptin plasma concentrations. The highest rate (K(in)) of [(125)I]leptin uptake was into the choroid plexuses (307.7+/-68.0 microl/min/g); however, this was not reflected in the CSF (8.9+/-4.1 microl/min/g) and indicates that this tissue tightly regulates leptin distribution. The multiple-time brain uptake of [(125)I]leptin was non-linear and suggested leptin could also be removed from the CNS. Studies using the incubated rat choroid plexus model found that [(125)I]leptin could cross the apical membrane of the choroid plexus to leave the CSF. However, this movement was not sensitive to unlabelled human leptin or specific transport inhibitors/modulators (including probenecid, digoxin, deltorphin II, progesterone and indomethacin).This study supports the concept of brain-barrier regulation of leptin distribution to the CNS, and highlights an important link between leptin and the cerebellum.

The effects of keratinocyte growth factor in preclinical models of mucositis.

The epithelium of the oral cavity and small intestine of the gastrointestinal tract have a high rate of cell renewal and as such, are sensitive to cytotoxic therapies that kill rapidly dividing cells. Mucositis is a complication of cancer therapy where impairment of the regenerative capacity of the epithelium leads to atrophy, ulceration and a loss of barrier function. Keratinocyte growth factor (KGF) is an epithelial cell-specific growth and differentiation factor that is trophic for the mucosal epithelium of the gastrointestinal tract. In this study, KGF in normal animals caused epithelial thickening in the squamous epithelium of the oral cavity and increased crypt depth and villus height of the small intestine. It also appeared to regulate gene expression in these tissues including that of some antioxidant enzymes and intestinal trefoil protein. KGF has been shown to be efficacious in several preclinical models of mucositis where KGF pretreatment reduced weight loss typically seen during and after the course of therapy and significantly improved survival. At a tissue level KGF reduced atrophy, accelerated regrowth, and decreased ulcer formation of the oral epithelium after irradiation, and improved crypt survival and prevented villus atrophy in the small intestine of irradiated or chemotherapy-treated mice. Preliminary studies suggest that its efficacy may be partly a consequence of the growth and differentiation effect, and also partly due to regulation of the expression of genes that play a role in mucosal protection. These data suggest that KGF may be useful for the prevention or treatment of mucositis in patients treated with regimens of cancer therapy that have gastrointestinal toxicity.

The effect of keratinocyte growth factor on healing of manifest radiation ulcers in mouse tongue epithelium.

The purpose of this study was to examine the effect of recombinant human keratinocyte growth factor (rHuKGF) on clinically manifest acute oral mucositis. The animal model utilized in this investigation was ventral tongue epithelium of C3H/Neu mice. In a first experiment, graded single doses were applied in order to define dose effect and time course of acute mucosal ulceration, as a clinically relevant endpoint. Irradiation was given to a 3 x 3 mm2 test field in the centre of the ventral tongue with 25 kV X-rays. A single dose of 18 Gy, i.e. a dose after which ulceration is expected in more than 99% of the animals, was applied in subsequent experiments. In the study group of 20 animals, rHuKGF was applied at a daily dose of 5 mg/kg subcutaneously from the time of first diagnosis of ulcer for a maximum of 5 days. In the control group, phosphate-buffered saline was used as a placebo. The time course of ulceration, i.e. individual ulcer duration, was analysed in both the control group without rHuKGF and the study group. Irradiation with graded single doses yielded an ED50 of 11.5 +/- 0.7 Gy (logit analysis). In responding animals, the latent time to first diagnosis of ulceration and the individual ulcer duration were independent of dose. Mean latency (+/- standard deviation) was 10.5 +/- 0.5 days, mean ulcer duration 3.9 +/- 0.6 days for doses 11, 13 and 16 Gy. After a dose of 18 Gy, 39 animals developed ulceration after a mean latency of 9.3 +/- 0.3 days (control and KGF-treated). The average ulcer duration was 4.2 +/- 0.9 days in the placebo group and 4.8 +/- 0.8 days in the KGF group (P = 0.02). We conclude that when rHuKGF treatment is delayed until radiation-induced ulcers are manifest, the therapeutic activity previously reported with other treatment schedules was not observed and there was a slight prolongation of duration of ulceration. These data suggest that during tumour radiotherapy, effective rHuKGF therapy schedules should include administration before the onset of ulcerative mucositis.

Positron emission tomography shows that intrathecal leptin reaches the hypothalamus in baboons.

Human obesity may be caused by a resistance to circulating leptin. Evidence from rodents and humans suggests that a major component of this resistance is an impairment in the ability of the blood-brain barrier (BBB) to transport leptin from the blood to the brain. One potential way to bypass the BBB is by administering leptin into the intrathecal (i.t.) space. To be effective, i.t. leptin would have to move caudally from the site of injection, enter the cranium, and reach the hypothalamic arcuate nucleus at the base of the pituitary fossa. However, many substances, especially small, lipid-soluble molecules, do not diffuse far from the site of i.t. injection but are resorbed back into blood. To determine whether i.t. leptin can move caudally, we injected leptin conjugated to diethylenetriaminepentaacetic acid (DTPA) and labeled with (68)Ga (G-Ob) into the lumbar space of three baboons. We also studied unconjugated DTPA labeled with (68)Ga, which did not move up the spinal cord but rapidly appeared in blood after i.t. injection. In contrast, G-Ob steadily moved toward the cranium and had reached the hypothalamus 91 and 139 min after i.t. injection in two baboons. We estimated the concentration of leptin in the hypothalamic region to be at least 8 ng/ml, which is about 40 times higher than cerebrospinal fluid levels in normal weight humans and about 4 times higher than the highest level ever recorded after the peripheral administration of leptin. In a third baboon, the leptin neither moved caudally nor appeared in the blood. We conclude that leptin administered i.t. can reach the hypothalamus in therapeutic concentrations, although there is considerable individual variation.

Regulation of glutathione redox status in lung and liver by conditioning regimens and keratinocyte growth factor in murine allogeneic bone marrow transplantation.

BACKGROUND: Reactive oxygen species (ROS) and glutathione (GSH) depletion contribute to organ injury after bone marrow transplantation (BMT). Keratinocyte growth factor (KGF) ameliorates graft-versus-host disease (GVHD)-associated organ injury in murine BMT models. METHODS: B10.BR mice received total body irradiation (TBI; day -1) +/- cyclophosphamide (Cy; 120 mg/kg/day i.p., days -3 and -2), then were transplanted on day 0 with C57BL/6 bone marrow + spleen cells as a source of GVHD-causing T cells. KGF (5 mg/kg/day subcutaneously [s.c.]) or saline was given on days -6, -5, and -4. Lung and liver GSH and oxidized GSH disulfide (GSSG) levels were measured on days 0 and 5 and glutathione redox potential (Eh) calculated. Organ malondialdehyde (MDA) was determined on day 5 as an index of ROS-mediated lipid peroxidation. RESULTS: In lung, TBI+BMT oxidized GSH Eh and increased MDA. Cy further oxidized lung GSH Eh. In liver, neither BMT regimen altered GSH redox status or MDA. KGF prevented the decrease in lung GSH after TBI+Cy and decreased lung MDA after both TBI and TBI+Cy. KGF increased liver GSH levels and GSH Eh after TBI and GSH Eh after TBI+Cy. CONCLUSIONS: In murine allogeneic BMT, TBI oxidizes the lung GSH redox pool and Cy exacerbates this response by 5 days post-BMT. In contrast, liver GSH redox status is maintained under these experimental conditions. KGF treatment attenuates the Cy-induced decrease in lung GSH, decreases post-BMT lung lipid peroxidation, and improves liver GSH redox indices. KGF may have a therapeutic role to prevent or attenuate GSH depletion and ROS-mediated organ injury in BMT.

The effects of repeated doses of keratinocyte growth factor on cell proliferation in the cellular hierarchy of the crypts of the murine small intestine.

Keratinocyte growth factor (KGF) administered on a daily basis for 3 or more days can result in dramatic changes in tissue architecture, particularly the thickness in oral epithelia, and can afford protection against the cytotoxic effects of radiation on the clonogenic stem cells in the crypts. This protection of intestinal stem cells (increased numbers of surviving crypts) is reflected in an increased survival of animals exposed to a lethal dose of irradiation. The mechanisms underlying these effects are not clear. The present experiments were designed to investigate the nature of any proliferative changes induced in the crypts of the small intestine by protracted exposure to KGF. Tritiated thymidine or bromodeoxyuridine labeling showed statistically significant increases in labeling in the stem cell zone of the crypt, with a concomitant reduction in labeling in the upper regions of the crypt corresponding to the late-dividing transit population. The increase in labeling in the lower regions of the crypt was also observed with Ki-67 staining, but the reduction in the upper regions of the crypt seen with tritiated thymidine was not observed with Ki-67. Metaphase arrest data suggest that the rate of progression through the cell cycle is essentially the same in KGF-treated animals as in controls, but there is a statistically significant increase in the number of mitotic events per crypt. Double labeling studies suggest that, at certain times of the day, there is a greater influx into S phase than efflux. The data overall indicate that KGF induces some complex proliferative changes in the intestinal crypts and are consistent with the hypothesis that the radioprotection may be afforded, at least in part, by a KGF-induced increase in stem cell numbers and/or increases in the number of stem cells in the S phase of the cell cycle. This alteration in the homeostasis of the crypt is compensated for by a foreshortening of the dividing transit lineage.

Modification of oral mucositis by keratinocyte growth factor: single radiation exposure.

PURPOSE: The aim was to quantify the effect of recombinant human keratinocyte growth factor (rhKGF) on acute oral mucositis induced by a single radiation dose, simulating accidental radiation exposure. MATERIAL AND METHODS: Tongue epithelium of the C3H/Neu mouse was irradiated with graded single doses of 25 kV X-rays to a 3 x 3 mm2 area in the centre of the lower tongue surface. Acute mucosal ulceration, as a clinically relevant reaction, was used as the quantal endpoint for dose response analyses by probit analysis. As a secondary endpoint the time-course, i.e. time to first diagnosis of ulcer (latent time) and individual ulcer duration, was analysed. KGF was applied before, after or in combination before and after radiation exposure. RESULTS: Administration of KGF in all protocols resulted in a significant reduction of the incidence of oral mucosal ulceration, as illustrated by an increase in iso-effective dose from 10.9 to 24.9 Gy; the corresponding dose-modification factors ranged between 1.7 and 2.3. The effect was most pronounced when KGF was applied after irradiation. In all protocols where KGF was given after irradiation, a significant shortening of the latent time to ulceration from 11 to 6-8 days was observed. CONCLUSIONS: The mechanisms underlying the amelioration of the oral mucosal response to single-dose irradiation remain unclear. However, KGF represents a promising approach for the effective management of acute radiation reactions in oral, gastrointestinal and cutaneous epithelia after radiation exposure.

Leptin transport at the blood--cerebrospinal fluid barrier using the perfused sheep choroid plexus model.

Leptin is secreted by adipose tissue and thought to regulate appetite at the central level. Several studies have explored the central nervous system (CNS) entry of this peptide across the blood-brain and blood-cerebrospinal fluid (CSF) barriers in parallel, but this is the first to explore the transport kinetics of leptin across the choroid plexus (blood-CSF barrier) in isolation from the blood-brain barrier (BBB). This is important as the presence of both barriers can lead to ambiguous results from transport studies. The model used was the isolated Ringer perfused sheep choroid plexus. The steady-state extraction of [(125)I]leptin (7.5 pmol l(-1)) at the blood face of the choroid plexus was 21.1+/-5.7%, which was greater than extraction of the extracellular marker, giving a net cellular uptake for [(125)I]leptin (14.0+/-3.7%). In addition, trichloroacetic acid precipitable [(125)I] was detected in newly formed CSF, indicating intact protein transfer across the blood-CSF barrier. Human plasma concentrations of leptin are reported to be 0.5 nM. Experiments using 0.5 nM leptin in the Ringer produced a concentration of leptin in the CSF of 12 pM (similar to that measured in humans). [(125)I]Leptin uptake at the blood-plexus interface using the single-circulation paired tracer dilution technique (uptake in <60 s) indicated the presence of a saturable transport system, which followed Michaelis-Menten-type kinetics (K(m)=16.3+/-1.8 nM, V(max)=41.2+/-1.4 pmol min(-1) g(-1)), and a non-saturable component (K(d)=0.065+/-0.002 ml min(-1) g(-1)). In addition, secretion of new CSF by the choroid plexuses was significantly decreased with leptin present. This study indicates that leptin transport at the blood-CSF barrier is via saturable and non-saturable mechanisms and that the choroid plexus is involved in the regulation of leptin availability to the brain.

Keratinocyte growth factor facilitates alloengraftment and ameliorates graft-versus-host disease in mice by a mechanism independent of repair of conditioning-induced tissue injury.

We have previously shown that pretreatment of mice with keratinocyte growth factor (KGF), an epithelial tissue repair factor, can ameliorate graft-versus-host disease (GVHD) after intensive chemoradiotherapeutic conditioning and allogeneic bone marrow transplantation (BMT). To determine whether this effect was dependent on a KGF-mediated mechanism affecting repair of conditioning-induced epithelial cell injury, we studied GVHD in the absence of conditioning using BALB/c severe combined immune-deficient (SCID) recipients given C57BL/6 T cells. KGF (5 mg/kg per day, subcutaneously) given either before or after T-cell transfer enhanced body weights and extended survival. KGF-treated recipients had elevated serum levels of the Th2 cytokine interleukin 13 (IL-13) on day 6 after T-cell transfer concomitant with reduced levels of the inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon gamma (IFN-gamma). A 3-day KGF pretreatment also depressed the secondary in vitro mixed lymphocyte response (MLR) of C57BL/6 splenocytes taken 7 days after in vivo alloimmunization with irradiated BALB/c spleen cells. To determine whether KGF would inhibit host-antidonor-mediated BM rejection, pan-T-cell-depleted BALB/c BM cells were infused into sublethally irradiated C57BL/6 mice and administered KGF either before or before and after BMT. Surprisingly, all KGF schedules tested actually resulted in enhanced alloengraftment. The presence of KGF receptor on donor antihost alloreactive T cells could not be detected by binding studies with radiolabeled KGF, reverse transcriptase-polymerase chain reaction, and Western blotting. Therefore, the mechanism of action of KGF on inhibiting T-cell-mediated immune effects may not be due to a direct effect of KGF on T cells. These studies demonstrate that KGF, by mechanisms independent of repair of conditioning-induced injury, has great potential as an anti-GVHD therapeutic agent with the added benefit of inhibiting the rejection of pan-T-cell-depleted donor BM allografts. (Blood. 2000;96:4350-4356)

Cyclophosphamide prevents systemic keratinocyte growth factor-induced up-regulation of surfactant protein A after allogeneic transplant in mice.

We reported that systemic keratinocyte growth factor (KGF) given before bone marrow transplantation (BMT) prevents allogeneic T cell-dependent lung inflammation assessed on Day 7 post-BMT, but the antiinflammatory effects of KGF were impaired in mice injected with both T cells and conditioning regimen of cyclophosphamide (Cy). Intratracheal KGF is known to stimulate the expression of surfactant protein A (SP-A), an oxidant-sensitive T cell immunomodulator produced by alveolar type II cells. We hypothesized that systemic KGF up-regulates SP-A after allogeneic BMT, and the addition of Cy may interfere with the ability of KGF to enhance SP-A production. The subcutaneous administration of recombinant human KGF (5 mg/kg on Days -6, -5, and -4 pre-BMT) increased SP-A protein and mRNA in allogeneic T cell-recipient irradiated mice measured on Day 7 post-BMT. In contrast, the same KGF treatment in irradiated mice given T cells and Cy failed to up-regulate SP-A mRNA and protein expression. In mixed lymphocyte reaction experiments designed to simulate the in vivo model, the addition of human SP-A (5-50 microg) to alloactivated T cells suppressed the production of interleukin-2 in a dose-dependent fashion. We conclude that the systemic pre-BMT injection of KGF in recipients of allogeneic T cells up-regulates SP-A, which may contribute to the early antiinflammatory effects of KGF. The protective KGF-mediated SP-A production is abolished in mice given alloreactive T cells plus Cy.

Induction of monocyte- and T-cell-attracting chemokines in the lung during the generation of idiopathic pneumonia syndrome following allogeneic murine bone marrow transplantation.

Idiopathic pneumonia syndrome (IPS) is a significant complication following bone marrow transplantation (BMT). We have developed a murine model in which severe IPS is induced by pre-BMT conditioning and allogeneic T cells and is characterized by the recruitment of host monocytes and donor T cells into the lung by day 7 post-BMT. Chemokines regulate cellular recruitment and the migration of cells into inflammatory lesions. In this study, we examined the profiles of chemokines produced locally in the lung (parenchyma and bronchoalveolar lavage fluid) and systemically (serum) during the generation of IPS in the peri-BMT period. Protein and messenger RNA (mRNA) levels of CC chemokines (monocyte/lymphocyte attractants), especially monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, RANTES (regulated upon activation normal T-cell expressed and secreted), and C10, were preferentially induced in the lung by day 7 postallogeneic BMT. In addition, there was an increase in mRNA for IP-10 (a monocyte and Th1-cell chemoattractant). The CXC chemokines MIP-2 and KC, known neutrophil attractants, were moderately elevated. For the most part, these increases in chemokines were dependent on the coinfusion of allogeneic T cells with the BM inoculum. Ribonuclease protection assay and in situ hybridization analyses post-BMT showed that the lung was a major producer of MCP-1, a potent inducer of monocyte chemotaxis. Increases in MCP-1 levels in the lung preceded host APC influx whereas MIP-1alpha levels accompanied donor T-cell infiltration. In summary, we have shown that monocyte- and T-cell-attracting chemokines are associated with monocyte and T-cell recruitment during IPS.

Partial saturation and regional variation in the blood-to-brain transport of leptin in normal weight mice.

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.

Enteral nutrition and keratinocyte growth factor regulate expression of glutathione-related enzyme messenger RNAs in rat intestine.

BACKGROUND: Malnutrition is associated with increased reactive oxygen species (ROS) formation and depletion of the critical antioxidant glutathione (GSH) in the intestine. The malnutrition-induced decrease in gut GSH levels is prevented by recombinant keratinocyte growth factor (KGF) administration. We investigated whether enzymes that are induced by oxidants and modulate tissue GSH supply are regulated by enteral nutrients or KGF at the messenger RNA (mRNA) level. METHODS: Adult rats were fasted for 3 days alone or fasted for 3 days then refed ad libitum. In a second model, rats were fasted for 3 days and then refed ad libitum or 25% of ad libitum intake with daily intraperitoneal saline or recombinant KGF (5 mg/kg/d) for 3 subsequent days. mRNA levels for gamma-glutamylcysteine synthetase (gamma-GCS), gamma-glutamyl transpeptidase (gamma-GT), glutathione-S-transferase Ya-subunit, gastrointestinal glutathione peroxidase (GI-GPx), and non-selenium-dependent glutathione peroxidase (ns-GPx) were determined in ileum and colon by ribonuclease protection assay. RESULTS: Fasting increased ileal gamma-GCS, ns-GPx, and glutathione-S-transferase mRNAs (by 36%, 165%, and 130% of controls) and decreased GI-GPx mRNA (to 55% of controls). In the colon, mRNAs for GSH-related enzymes were unchanged by fasting or refeeding. Prolonged enteral nutrient restriction (25% refeeding after a 3-day fast) increased gamma-GCS and glutathione-S-transferase mRNAs (by >270% of controls), decreased GI-GPx mRNA (to <50% of controls) in ileum and colon and increased ns-GPx mRNA (by 180%) in colon. KGF treatment increased ns-GPx mRNA in the ileum and colon and glutathione-S-transferase mRNA in the colon (by >200% of controls). CONCLUSIONS: Enteral nutrient intake regulates GSH-related enzyme mRNA levels in the intestine, which may contribute to the decrease in mucosal GSH during malnutrition. Increased ns-GPx and glutathione-S-transferase mRNA levels during malnutrition and with KGF administration may increase detoxifying functions in the gut under these conditions.

KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT.

We investigated keratinocyte growth factor (KGF) as a pretreatment therapy for idiopathic pneumonia syndrome (IPS) generated as a result of lung damage and allogeneic T cell-dependent inflammatory events occurring in the early peri-bone marrow (BM) transplant (BMT) period. B10.BR (H2(k)) recipient mice were transplanted with C57BL/6 (H2(b)) BM with spleen cells after lethal irradiation with and without cyclophosphamide conditioning with and without subcutaneous KGF pretreatment. KGF-pretreated mice had fewer injured alveolar type II (ATII) cells at the time of BMT and exhibited ATII cell hyperplasia at day 3 post-BMT. The composition of infiltrating cells on day 7 post-BMT was not altered by KGF pretreatment, but the frequencies of cells expressing the T-cell costimulatory molecules B7.1 and B7.2 and mRNA for the cytolysin granzyme B (usually increased in IPS) were decreased by KGF. Sera from KGF-treated mice had increases in the Th2 cytokines interleukin (IL)-4, IL-6, and IL-13 4 days after cessation of KGF administration (i.e., at the time of BMT). These data suggest that KGF hinders IPS by two modes: 1) stimulation of alveolar epithelialization and 2) attenuation of immune-mediated injury as a consequence of failure to upregulate cytolytic molecules and B7 ligand expression and the induction of anti-inflammatory Th2 cytokines in situ.

Differential regulation of leptin transport by the choroid plexus and blood-brain barrier and high affinity transport systems for entry into hypothalamus and across the blood-cerebrospinal fluid barrier.

Leptin is a circulating hormone that controls food intake and energy homeostasis. Little is known about leptin entry into the central nervous system (CNS). The blood-cerebrospinal fluid (CSF) barrier at the choroid plexus and the blood-brain barrier (BBB) at the cerebral endothelium are two major controlling sites for entry of circulating proteins into the brain. In the present study, we characterized leptin transport across the blood-CSF barrier and the BBB by using a brain perfusion model in lean rats. Rapid, high-affinity transport systems mediated leptin uptake by the hypothalamus (KM = 0.2 ng/ml) and across the blood-CSF barrier (KM = 1.1 ng/ml). High affinity in vivo binding of leptin was also detected in the choroid plexus (KD = 2.6 ng/ml). In contrast, low affinity carriers for leptin (KM = 88 to 345 ng/ml) were found at the BBB in the CNS regions outside the hypothalamus (e.g. cerebral cortex, caudate nucleus, hippocampus). Our findings suggest a key role of high affinity leptin transporters in the hypothalamus and choroid plexus in regulating leptin entry into the CNS and CSF under physiological conditions. Low affinity transporters at the BBB outside the hypothalamus could potentially contribute to overall neuropharmacological effects of exogenous leptin.

Regulation of keratinocyte growth factor (KGF) and KGF receptor mRNAs by nutrient intake and KGF administration in rat intestine.

The aim of this study was to investigate the regulation of keratinocyte growth factor (KGF) and KGF receptor mRNAs by diet and KGF treatment in rat intestine. Fasting for three days up-regulated KGF and KGF receptor mRNA levels in ileum and increased KGF receptor mRNA expression in colon. KGF and KGF receptor mRNA levels returned toward control values with ad libitum refeeding but remained elevated when refeeding was limited to 25% of ad libitum intake. KGF treatment during nutrient repletion did not alter intestinal KGF mRNA levels but increased KGF receptor mRNA abundance in ileum and colon. We conclude that the increase in KGF and KGF receptor mRNAs induced by malnutrition may be an adaptive response to attenuate gut mucosal atrophy in this setting. The gut-trophic effects of KGF treatment may be mediated, in part, by up-regulation of the KGF receptor mRNA in small bowel and colon.

Keratinocyte growth factor enhances early gut adaptation in a rat model of short bowel syndrome.

OBJECTIVE: To evaluate the effects of keratinocyte growth factor (KGF) on intestinal adaptation after resection of 85% of the small intestine and consider its potential application in short bowel syndrome (SBS). STUDY DESIGN: Experimental study using a known model of SBS. ANIMAL POPULATION: Thirty male Sprague Dawley rats. METHODS: Four groups of animals were designated. Two groups underwent 85% resection of the small intestine, while the other two groups were sham-operated, undergoing transection and reanastomosis. Resected and sham-operated groups then received either 3 mg/kg KGF or vehicle subcutaneously daily for 3 days. Gut adaptation was evaluated by measurements of mucosal cellularity and biochemical activity in duodenal, jejunal, and ileal segments. RESULTS: Significant small intestinal growth after bowel resection alone was confirmed in resected versus sham-operated rats. KGF further augmented this growth in the resected animals. Mucosal wet weight of the small intestine increased with resection and was further increased (by 20% or more) with KGF administration. Mucosal thickness, villus length, and crypt depth exhibited similar patterns of response. The KGF-induced increase in mucosal morphology was accompanied by increased mucosal DNA and protein content, followed by a trend toward increased mucosal enzyme activity. Histology demonstrated an increase in goblet cells in KGF-treated animals. In situ hybridization analysis demonstrated that KGF markedly increased mucosal expression of intestinal trefoil protein (ITF) mRNA. CONCLUSIONS: KGF enhances gut growth, differentiation, and gene regulation during adaptation in rat small intestine after massive resection. CLINICAL RELEVANCE: KGF may be beneficial in the management of veterinary and human patients undergoing massive intestinal resection.

Mortality and kidney histopathology of chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains.

An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 x 10(3) or 1 x 10(6) bacteria fish(-1), or by a 24 h immersion in 1 x 10(5) or 1 x 10(7) bacteria ml(-1). For 22 wk fish were held in 12 degrees C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73 %). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.

Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene msa in virulent and attenuated strains of Renibacterium salmoninarum.

Virulence mechanisms utilized by the salmonid fish pathogen Renibacterium salmoninarum are poorly understood. One potential virulence factor is p57 (also designated MSA for major soluble antigen), an abundant 57 kDa soluble protein that is predominately localized on the bacterial cell surface with significant levels released into the extracellular milieu. Previous studies of an attenuated strain, MT 239, indicated that it differs from virulent strains in the amount of surface-associated p57. In this report, we show overall expression of p57 in R. salmoninarum MT 239 is considerably reduced as compared to a virulent strain, ATCC 33209. The amount of cell-associated p57 is decreased while the level of p57 in the culture supernatant is nearly equivalent between the strains. To determine if the lowered amount of cell-associated p57 was due to a sequence defect in p57, a genetic comparison was performed. Two copies of the gene encoding p57 (msa1 and msa2) were found in 33209 and MT 239, as well as in several other virulent isolates. Both copies from 33209 and MT 239 were cloned and sequenced and found to be identical to each other, and identical between the 2 strains. A comparison of msa1 and msa2 within each strain showed that their sequences diverge 40 base pairs 5' to the open reading frame, while sequences 3' to the open reading frame are essentially identical for at least 225 base pairs. Northern blot analysis showed no difference in steady state levels of msa mRNA between the 2 strains. These data suggest that while cell-surface localization of p57 may be important for R. salmoninarum virulence, the differences in localization and total p57 expression between 33209 and MT 239 are not due to differences in msa sequence or differences in steady state transcript levels.

Impaired transport of leptin across the blood-brain barrier in obesity.

Leptin is a 17-kDa protein secreted by fat cells that regulates body adiposity by crossing the blood-brain barrier (BBB) to affect feeding and thermogenesis. Obese human and rodent models of dietary obesity have shown decreased sensitivity to blood-borne leptin, postulated to be due to impaired transport of leptin across the BBB. We show here that the transport rate of leptin across the BBB is reduced about 2/3 in 12-month-old obese CD-1 mice. In a follow-up study, a perfusion method was used that replaced the blood with a buffer containing low concentrations of radioactive leptin. Obese mice still had lower rates of transport into the brain than lean mice, which shows that the reduction in transport rate associated with obesity is not due simply to saturation of transporter secondary to higher serum leptin levels as has been thought, but to a decreased capacity of the BBB to transport leptin. This suggests a new model for obesity in which a defect in the BBB transport of leptin into the CNS underlies the insensitivity to leptin and leads to obesity.