Ischemia-reperfusion increases transfection efficiency of intracoronary adenovirus type 5 in pig heart in situ.

Efficiency of intracoronary (IC) adenoviral vector transfection is impaired by the vascular endothelium. Ischemia and substances that increase vascular permeability (sodium nitroprusside, nitroglycerin) may augment adenoviral vector transfection efficiency (TE). We tested whether TE of adenoviral vector following IC infusion is improved by nitrates or by ischemia. Fluoroscopically guided angioplasty balloon catheters occluded the coronary artery in Yorkshire pigs and delivered adenoviral type 5 vector encoding the luciferase gene (Ad5Luc, 10(11) viral particles). TE (luciferase activity) was minimal and was not augmented by IC co-administration of 50 µg/min sodium nitroprusside to nonischemic myocardium. Two (but not one) 3-min episodes of occlusion tended to increase luciferase activity (p=0.06), and luciferase activity was further increased by IC co-administration of nitroglycerin (p<0.001). After 75 min of coronary artery occlusion, luciferase activity was greater than with shorter periods of ischemia, and was significantly greater in the ischemia-reperfused zone compared to the border zone 3 and 14 days after infusion; there was no transfection in nonischemic myocardium. IC delivery of Ad5Luc into post-ischemic myocardium caused no local inflammation or hemodynamic instability. We conclude that the uptake of IC Ad5 to ischemic reperfused myocardium validates use of IC Ad5 delivery protocols in future human gene therapy trials in patients following myocardial ischemia.

Enigmol: a novel sphingolipid analogue with anticancer activity against cancer cell lines and in vivo models for intestinal and prostate cancer.

Sphingoid bases are cytotoxic for many cancer cell lines and are thought to contribute to suppression of intestinal tumorigenesis in vivo by ingested sphingolipids. This study explored the behavior of a sphingoid base analogue, (2S,3S,5S)-2-amino-3,5-dihydroxyoctadecane (Enigmol), that cannot be phosphorylated by sphingosine kinases and is slowly N-acylated and therefore is more persistent than natural sphingoid bases. Enigmol had potential anticancer activity in a National Cancer Institute (NCI-60) cell line screen and was confirmed to be more cytotoxic and persistent than naturally occurring sphingoid bases using HT29 cells, a colon cancer cell line. Although the molecular targets of sphingoid bases are not well delineated, Enigmol shared one of the mechanisms that has been found for naturally occurring sphingoid bases: normalization of the aberrant accumulation of ß-catenin in the nucleus and cytoplasm of colon cancer cells due to defect(s) in the adenomatous polyposis coli (APC)/ß-catenin regulatory system. Enigmol also had antitumor efficacy when administered orally to Min mice, a mouse model with a truncated APC gene product (C57Bl/6J(Min/+) mice), decreasing the number of intestinal tumors by half at 0.025% of the diet (w/w), with no evidence of host toxicity until higher dosages. Enigmol was also tested against the prostate cancer cell lines DU145 and PC-3 in nude mouse xenografts and suppressed tumor growth in both. Thus, Enigmol represents a novel category of sphingoid base analogue that is orally bioavailable and has the potential to be effective against multiple types of cancer.

Pathogenic virus-specific T cells cause disease during treatment with the calcineurin inhibitor FK506: implications for transplantation.

Recently, several cases of fatal lymphocytic choriomeningitis virus (LCMV) infection occurred in transplant recipients being treated with the immunosuppressive calcineurin inhibitor FK506. These findings were surprising because LCMV is a noncytolytic virus. To understand how a noncytolytic virus can cause disease under conditions of immunosuppression, we used the mouse LCMV model and found that, similar to the observations in human transplant recipients, LCMV infection of FK506-treated mice resulted in a lethal disease characterized by viremia, lack of seroconversion, and minimal lymphocytic infiltrates in the tissues. However, despite the apparent absence of an antiviral immune response, this disease was orchestrated by virus-specific T cells. FK506 did not prevent the generation and proliferation of LCMV-specific T cells but instead altered their differentiation so that these effector T cells lost the ability to control virus but were still capable of mediating disease. These pathogenic T cells initiated a cytokine storm characterized by high levels of tumor necrosis factor (TNF) and interleukin 6 (IL-6), and depletion of T cells or blockade of these inflammatory cytokines prevented the lethal disease. Our study shows that inhibiting calcineurin can generate pathogenic T cells and indicates that T cell-mediated viral disease can occur even under conditions of immunosuppression. Furthermore, we identify a potential strategy (blockade of TNF and IL-6) for treatment of transplant recipients who have acute complications of viral infection.

PIKE-A is required for prolactin-mediated STAT5a activation in mammary gland development.

PI 3-kinase enhancer A (PIKE-A) is critical for the activation of Akt signalling, and has an essential function in promoting cancer cell survival. However, its physiological functions are poorly understood. Here, we show that PIKE-A directly associates with both signal transducer and activator of transcription 5a (STAT5a) and prolactin (PRL) receptor, which is essential for PRL-provoked STAT5a activation and the subsequent gene transcription. Depletion of PIKE-A in HC11 epithelial cells diminished PRL-induced STAT5 activation and cyclin D1 expression, resulting in profoundly impaired cell proliferation in vitro. To confirm the function of PIKE-A in PRL signalling in vivo, we generated PIKE knockout (PIKE-/-) mice. PIKE-/- mice displayed a severe lactation defect that was characterized by enhanced apoptosis and impaired proliferation of mammary epithelial cells. At parturition, STAT5 activation and cyclin D1 expression were substantially reduced in the mammary epithelium of PIKE-/- mice. The defective mammary gland development in PIKE-/- mice was rescued by overexpression of a mammary-specific cyclin D1 transgene. These data establish a critical function for PIKE-A in mediating PRL functions.

Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of citrobacter rodentium.

We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals. Here, we report that oral infection of C57BL/6 mice with C. rodentium, which produced only mild clinical signs and symptoms, produced very similar effects on hepatic P450 expression in this strain. As in HeOu mice, CYP4A mRNAs and proteins were among the most sensitive to down-regulation, whereas CYP4F18 was induced. CYP2D9 mRNA was also induced 8- to 9-fold in the C57BL/6 mice. The time course of P450 regulation followed that of colonic inflammation and bacterial colonization, peaking at 7 to 10 days after infection and returning to normal at 15 to 24 days as the infection resolved. These changes also correlated with the time course of significant elevations in the serum of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, as well as of interferon-gamma and IL-2, with serum levels of IL-6 being markedly higher than those of the other cytokines. Intraperitoneal administration of C. rodentium produced a rapid down-regulation of P450 enzymes that was quantitatively and qualitatively different from that of oral infection, although CYP2D9 was induced in both models, suggesting that the effects of oral infection on the liver are not due to bacterial translocation.

Suppression of NF-kappaB activation blocks osteoclastic bone resorption during estrogen deficiency.

Postmenopausal osteoporosis stems from an imbalance in osteoclastic bone resorption with respect to osteoblastic bone formation, a consequence of estrogen deficiency. The nuclear factor-kappaB (NF-kappaB) signal transduction pathway is critical for osteoclast formation and resorption, and suppression of NF-kappaB activation has been shown to block bone resorption in vitro, and to ameliorate inflammatory bone loss in vivo. The use of NF-kappaB antagonists to blunt the bone loss associated with estrogen deficiency however, has not been previously reported. In this study, we investigated whether pharmacological suppression of NF-kappaB signaling protects mice against ovariectomy (ovx)-induced bone loss. Ovx mice were treated with the potent NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) for 4 weeks and bone mineral density (BMD) and indices of bone structure quantitated by dual-energy X-ray absorptiometry (DXA), and micro-computed tomography (microCT). In vivo indices of bone resorption were quantitated in mouse serum using the biochemical marker C-terminal telopeptide of collagen (CTx). Our data revealed that NF-kappaB suppression significantly prevented ovx-induced bone destruction by preventing the increase in ovx-induced osteoclastic bone resorption. Our data suggest that NF-kappaB inhibitors may represent novel anticatabolic therapeutic agents for the amelioration of postmenopausal bone loss.

Safingol toxicology after oral administration to TRAMP mice: demonstration of safingol uptake and metabolism by N-acylation and N-methylation.

Safingol [(2S,3S)-2-amino-1,3-octadecanediol] is an unnatural l-threo-stereoisomer of sphinganine that is cytotoxic for cancer cells in culture and is being tested in phase 1 human clinical trials. To determine if safingol can be absorbed orally and if it affects prostate cancer in a mouse strain used in prostate cancer studies, safingol was fed to TRAMP (transgenic adenocarcinoma of mouse prostate) mice for 2 weeks at 0.0125% to 0.1% w/w of the diet. Analysis of safingol and safingol metabolites in blood and tissues by liquid chromatography electrospray ionization tandem mass spectrometry revealed uptake in tissue and extensive conversion of safingol to N-acyl species (comparable to natural "ceramides") and mono-, di-, and tri-N-methyl metabolites that have not been observed previously. Safingol caused significant hepatotoxicity at all dosages, as reflected in elevated liver alanine aminotransferase, and at the highest dose (0.1 %) caused changes in liver histology (appearance of autophagosomal vacuoles) and renal toxicity (based on elevation of blood urea nitrogen) and decreases in packed blood cell volume and body weight. Safingol did not inhibit the prostate pre-neoplastic lesion (prostate intraepithelial neoplasia) in TRAMP mice; however, additional studies at lower dosages for longer time were not pursued due to host toxicity. Safingol and its N-methyl metabolites were cytotoxic to both a human prostate cell line (DU145) and mouse BALB 3T3 cells; therefore, the host and potential antitumor toxicity may be due to multiple molecular species of safingol.

Cytokine-mediated disruption of lymphocyte trafficking, hemopoiesis, and induction of lymphopenia, anemia, and thrombocytopenia in anti-CD137-treated mice.

CD137-mediated signals costimulate T cells and protect them from activation-induced apoptosis; they induce curative antitumor immunity and enhance antiviral immune responses in mice. In contrast, anti-CD137 agonistic mAbs can suppress T-dependent humoral immunity and reverse the course of established autoimmune disease. These results have provided a rationale for assessing the therapeutic potential of CD137 ligands in human clinical trials. In this study, we report that a single 200-mug injection of anti-CD137 given to otherwise naive BALB/c or C57BL/6 mice led to the development of a series of immunological anomalies. These included splenomegaly, lymphadenopathy, hepatomegaly, multifocal hepatitis, anemia, altered trafficking of B cells and CD8 T cells, loss of NK cells, and a 10-fold increase in bone marrow (BM) cells bearing the phenotype of hemopoietic stem cells. These events were dependent on CD8 T cells, TNF-alpha, IFN-gamma, and type I IFNs. BM cells up-regulated Fas, and there was a significant increase in the number of CD8+ T cells that correlated with a loss of CD19+ and Ab-secreting cells in the BM. TCR Valphabeta usage was random and polyclonal among liver-infiltrating CD8 T cells, and multifocal CD8+ T cell infiltrates were resolved upon termination of anti-CD137 treatment. Anti-CD137-treated mice developed lymphopenia, thrombocytopenia, and anemia, and had lowered levels of hemoglobin and increased numbers of reticulocytes.

Investigation of appropriate sanitization frequency for rodent caging accessories: evidence supporting less-frequent cleaning.

The Guide for the Care and Use of Laboratory Animals states that sanitization of caging accessories (for example, filter tops and wire-bar lids) should be done every 2 wk. In this study we tested the hypothesis that organic contamination measured by the presence of ATP associated with organic material (measured with luciferase test swabs) and the number of bacterial colony-forming units (as determined by use of replicate organism detection and counting plates) on caging accessories did not differ significantly at 2 wk versus several months of use. The study evaluated 4 groups: mouse and rat ventilated and static wire-bar cages with or without filter tops (n = 10 per group). The cages were evaluated at several time points from 2 wk to 6 mo. For every cage type, ATP levels did not differ significantly between 14 and 90 d and, in most cases, between 14 and 180 d. In addition the number of bacterial colonies did not differ significantly between 14 and 120 d (and, in some cases, between 14 and 180 d). This study provides data relevant to establishing a validated frequency for sanitization of rodent caging accessories while controlling, and potentially decreasing, costs associated with sanitization.

Use of broad-spectrum antimicrobials in the eradication of unknown aquatic pathogens in a zebrafish larval rearing system.

A zebrafish larval rearing system experienced a surge in mortality rates soon after the introduction of new stocks. A comprehensive water analysis of pH, nitrites, nitrates, ammonia, chlorine, carbonate hardness, general hardness, and conductivity identified no anomalies. Observations via light microscopy of affected fry revealed consistent signs of impaired mobility, blood clotting, and eventual heart hemorrhage resulting in the death of 90 to 100% of the fry by the age of 2 weeks. Collection of sufficient tissue samples for a histological investigation proved problematic due to the fry's diminutive size. Because a causal agent could not be isolated satisfactorily, the use of a broad-spectrum antibiotic was deemed necessary. After considering many broad-spectrum antibiotics for treatment, we implemented a two-tiered approach for treatment. The rearing system was treated with a nitrofurazone derivative, whereas the adult populations were treated using multi-antibiotic food pellets. The rearing system was treated for 3 weeks, and the adult population was treated for 2 weeks. After the completion of the antibiotic treatments, the biological filters of all of the medicated systems were seeded with nitrifying bacterial cultures. Upon the maturation of the rearing systems' biological filters, mortality rates returned to pre-outbreak levels. There have been no re-occurrences of the fish mortality since the completion of treatment. This epidemic provided some valuable lessons, lessons that if followed, will ensure faster response to unknown pathogens in the future.

Urolithiasis associated with experimental lymphocytic choriomeningitis virus inoculation in Lewis rats.

A high frequency of struvite urolithiasis, hydronephrosis, and other urinary tract lesions developed in a group of Lewis rats inoculated intracranially with lymphocytic choriomeningitis virus (LCMV). Initially, clinically ill rats were referred to necropsy: 30 rats over 3 years. These rats had high frequency of urolithiasis (8/30, 27%), hydronephrosis (12/30, 40%), cystitis (9/30, 30%), transitional cell carcinoma (4/30, 13%), and pyelonephritis (19/30, 63%). Lesions were more common in LCMV-inoculated rats. After this trend was noted, all rats on this protocol were necropsied as part of a cohort study (n = 144). Although the apparent frequency of disease was lower due to increased sampling, there still was a high number of urolithiasis (9/144, 6%) and hydronephrosis (40/144, 28%) cases. All cases of urolithiasis developed in rats inoculated with LCMV (9/44, 20%), as did most cases of hydronephrosis (31/44, 70%). Although sham-injected and uninoculated control rats also had high frequency of hydronephrosis (6/57 [11%] and 3/43 [7%], respectively), LCMV-inoculated rats had a significantly higher frequency of disease than did sham inoculated (P < 0.0001) and uninoculated (P < 0.0001) controls. These results suggest that Lewis rats may be predisposed to developing lesions of the urinary tract, and that intracranial inoculation of rats with LCMV augments this tendency, leading to formation of struvite calculi and associated urinary tract disease.

Dietary soy sphingolipids suppress tumorigenesis and gene expression in 1,2-dimethylhydrazine-treated CF1 mice and ApcMin/+ mice.

Dietary supplementation with milk sphingolipids inhibits colon tumorigenesis in CF1 mice treated with a colon carcinogen [1,2-dimethylhydrazine (DMH)] and in multiple intestinal neoplasia (Min) mice, which develop intestinal tumors spontaneously. Plant sphingolipids differ structurally from those of mammals [soy glucosylceramide (GlcCer) consists predominantly of a 4,8-sphingadiene backbone and alpha-hydroxy-palmitic acid], which might affect their bioactivity. Soy GlcCer was added to the AIN-76A diet (which contains <0.005% sphingolipid) to investigate whether it would also suppress tumorigenesis in these mouse models. Soy GlcCer reduced colonic cell proliferation in the upper half of the crypts in mice treated with DMH by 50 and 56% (P < 0.05) at 0.025 and 0.1% of the diet (wt/wt), respectively, and reduced the number of aberrant colonic crypt foci (an early marker of colon carcinogenesis) by 38 and 52% (P < 0.05). Min mice fed diets containing 0.025 and 0.1% (wt/wt) soy GlcCer developed 22 and 37% fewer adenomas (P < 0.05), respectively. The effects of dietary sphingolipids on gene expression in the intestinal mucosal cells of Min mice were analyzed using Affymetrix GeneChip microarrays. Soy GlcCer affected the expression of 96 genes by > or = 2-fold in a dose-dependent manner, increasing 32 and decreasing 64. Decreases in the mRNA expression of two transcription factors associated with cancer, hypoxia-induced factor 1 alpha (HIF1 alpha) and transcription factor 4 (TCF4), were confirmed by quantitative RT-PCR. In conclusion, soy GlcCer suppressed colon tumorigenesis in two mouse models; hence, plant sphingolipids warrant further investigation as inhibitors of colon cancer. Because soy contains relatively high amounts of GlcCer, sphingolipids may partially account for the anticancer benefits attributed to soy-based foods.

Ectopical expression of human MUC18 increases metastasis of human prostate cancer cells.

MUC18, a cell adhesion molecule (CAM), has been reported to be a diagnostic marker for the early detection of the metastatic potential of prostate cancers as well as implicated to be an important determinant for mediating the tumorigenesis and metastasis of prostate cancer. To test the hypothesis, we further investigated the possible role of MUC18 in the malignant progression of human prostate cancer. The human MUC18-minus, non-metastatic human prostate cancer LNCaP cells were transfected with the human cytomegalovirus immediate-early gene (HCMV-IE) promoter-driven human MUC18 (huMUC18) cDNA. The G418-resistant (G418R)-LNCaP clones that expressed a high level of huMUC18 were selected and used for testing the effect of huMUC18 expression on the in vitro growth, motility, and invasiveness as well as on the in vivo metastasis (via orthotopical injection) in a xenograft nude mouse model. HuMUC18 expression increased by four- to fivefold of in vitro motility and invasiveness of LNCaP cells. Anti-huMUC18 antibody significantly inhibited the in vitro motility and invasiveness of huMUC18-expressing LNCaP clones, but not the control clones. We suggest that huMUC18 expression is responsible for increasing these behaviors of LNCaP cells. HuMUC18 expression also directly increased the in vivo metastatic abilities of the LNCaP cells from the prostate gland to multiple distant organs. Western blot and immunohistochemistry analyses showed that the prostatic tumors as well as metastatic lesions expressed high levels of MUC18, indicating that they originated from the injected huMUC18-expressing LNCaP cells. We therefore conclude that HuMUC18 is an important determinant in increasing metastasis of human prostate cancer LNCaP cells to distant organs in a nude mouse model.

Cutaneous acariasis in the African clawed frog (Xenopus laevis).

Increased mortality was observed in a single colony of 50 Xenopus laevis. The frogs were used as oocyte donors in developmental biology studies. Necropsy findings included dermal erythema and petechiation consistent with red leg syndrome; dermal ulcerations and white, filamentous growths on the skin were consistent with Saprolegnia sp. Microscopic evaluation of the skin and fungus revealed an astigmatid mite similar to those of the genus Rhizoglyphus. The mite was also found in the water and the biological filter of the tanks housing the frogs. This mite is considered not to be a parasite of X. laevis; instead, it feeds off moss, fungi, and detritus. Subsequent evaluation of the sphagnum moss used for shipping the frogs from the supplier revealed the same mite in the moss. Our hypothesis is that the mite was introduced into the tank with the shipment of new frogs in sphagnum moss. The mites lived within the biological filter, and were only found after the growth of Saprolegnia sp. attracted the mites to the frogs. Laboratory animal care and veterinary personnel should consider non-pathogenic species of mites in the differential diagnosis of acariasis in Xenopus frogs.

Sphingomyelin in the suppression of colon tumors: prevention versus intervention.

Intestinal cells are regularly exposed to sphingolipid metabolites, i.e., ceramide and sphingoid bases, after hydrolysis of complex sphingolipids from the diet. These metabolites are known regulators of cell growth, differentiation, and death. Non-pharmacological amounts in the diet have been shown to inhibit early stages of chemically induced colon cancer in mice. To distinguish between chemopreventive and chemotherapeutic effects of sphingomyelin supplements, mice were fed sphingomyelin before and after tumor initiation. Both applications drastically reduced tumor formation, without a significant difference among the groups, indicating that sphingolipids are as effective in the chemoprevention of tumors as in early intervention. The normalization of cell proliferation and rate of apoptosis, but not the induction of differentiation, seem to be key players in the suppression of tumor formation by dietary sphingomyelin. This may have implications for the development of a cancer prevention or treatment strategy with sphingolipids as an alternative to conventional drugs.

Confirmed persistent mouse hepatitis virus infection and transmission by mice with a targeted null mutation of tumor necrosis factor to sentinel mice, using short-term exposure.

Mouse hepatitis virus (MHV) infection in immunocompetent mice is typically self limiting, and transmission is short lived. With the recent surge in the development of genetically engineered mutant mice with alterations in immune system components, however, MHV clearance may be disrupted. We report confirmed persistent transmission of MHV from tumor necrosis factor (TNF) knockout mice, B6.129S1-Tnftm1Lj (TNF -/-), to nude and immunocompetent sentinel mice over a period of five months. Infection with MHV was confirmed in nude sentinel mice by use of reverse transcriptase-polymerase chain reaction (RT-PCR) detection of viral RNA in ascending colon and feces. The RT-PCR-analyzed specimens recovered from sentinel animals were sequenced, and 92% homology to the N region of the MHV strain S genome was documented. In addition, immunocompetent mice had evidence of seroconversion to MHV infection and RT-PCR-positive fecal and ascending colon specimens after only 24 h of direct contact with the TNF -/- mice. To the authors' knowledge, this is the first reported experimental evidence that MHV transmission can occur for several months, from persistently infected mice to sentinel mice, over a short-term exposure period.

Postanesthesia death and suspected peracute endotoxic shock due to Pseudomonas putida in a cynomolgous macaque (Macaca fascicularis).

An adult male cynomolgous macaque (Macaca fascicularis) died suddenly after anesthesia for a positron emission tomography scan. Bacteriologic culture of the mucopurulent secretions recovered from the endotracheal tube yielded heavy growth of Pseudomonas putida, a known endotoxin producer. Histologically, the lungs had severe, diffuse perivascular edema and neutrophils marginating to the endothelium. The sudden death and the pathologic findings were consistent with peracute endotoxic shock. Numerous environmental swab specimens of the surgical suite and equipment were submitted for bacteriologic culture, as were swab specimens of endotracheal secretions from a control animal; however, Pseudomonas putida was not isolated from any specimen. The animal in this report may have carried Pseudomonas putida as a commensal in the oropharynx, and the stress of anesthesia may have resulted in increased sensitivity to the endotoxin.

Sphingoid bases and de novo ceramide synthesis: enzymes involved, pharmacology and mechanisms of action.

The sphingoid base backbones of sphingolipids (sphingosines, sphinganines, 4-hydroxysphinganines and others) are highly bioactive species directly and-in most cases-as their metabolites, the N-acyl-sphingoid bases (ceramides) and sphingoid base 1-phosphates. The complexity of these compounds affords many opportunities to prepare synthetic analogs for studies of sphingolipid metabolism and the functions of the sphingoid bases and metabolites. Described in this review are methods for the preparation of libraries of sphingoid bases, including a series of 1-deoxy-analogs, as well as information about their metabolism and biological activities. Findings with these compounds have uncovered some of the complications of working with compounds that mimic a naturally occurring biomodulator-such as that they are sometimes metabolized by enzymes that handle the endogenous compounds and the products may have potent (and unexpected) biological activities. Through studying such compounds, there is now a greater understanding of the metabolism and mechanism(s) of action of naturally occurring sphingoid bases as well as of these analogs.

A cure for murine sickle cell disease through stable mixed chimerism and tolerance induction after nonmyeloablative conditioning and major histocompatibility complex-mismatched bone marrow transplantation.

The morbidity and mortality associated with sickle cell disease (SCD) is caused by hemolytic anemia, vaso-occlusion, and progressive multiorgan damage. Bone marrow transplantation (BMT) is currently the only curative therapy; however, toxic myeloablative preconditioning and barriers to allotransplantation limit this therapy to children with major SCD complications and HLA-matched donors. In trials of myeloablative BMT designed to yield total marrow replacement with donor stem cells, a subset of patients developed mixed chimerism. Importantly, these patients showed resolution of SCD complications. This implies that less toxic preparative regimens, purposefully yielding mixed chimerism after transplantation, may be sufficient to cure SCD without the risks of myeloablation. To rigorously test this hypothesis, we used a murine model for SCD to investigate whether nonmyeloablative preconditioning coupled with tolerance induction could intentionally create mixed chimerism and a clinical cure. We applied a well-tolerated, nonirradiation-based, allogeneic transplantation protocol using nonmyeloablative preconditioning (low-dose busulfan) and costimulation blockade (CTLA4-Ig and anti-CD40L) to produce mixed chimerism and transplantation tolerance to fully major histocompatibility complex-mismatched donor marrow. Chimeric mice were phenotypically cured of SCD and had normal RBC morphology and hematologic indices (hemoglobin, hematocrit, reticulocyte, and white blood cell counts) without evidence of graft versus host disease. Importantly, they also showed normalization of characteristic spleen and kidney pathology. These experiments demonstrate the ability to produce a phenotypic cure for murine SCD using a nonmyeloablative protocol with fully histocompatibility complex-mismatched donors. They suggest a future treatment strategy for human SCD patients that reduces the toxicity of conventional BMT and expands the use of allotransplantation to non-HLA-matched donors.

Preclinical evaluation of a novel device for delivering brachytherapy to the margins of resected brain tumor cavities.

OBJECT: The objectives of this study were to evaluate the safety and performance of a new brachytherapy applicator in the treatment of resected brain tumors in a canine model. METHODS: The brachytherapy applicator is an inflatable balloon catheter that is implanted in the resection cavity remaining after a brain tumor has been debulked. After implantation the balloon is inflated with Iotrex, a sterile solution containing organically bound iodine-125. The low-energy photons emitted by the iodine-125 deposit a therapeutic radiation dose across short distances from the surface of the balloon. After delivery of a prescribed radiation dose to the targeted volume, the radioactive fluid is retrieved and the catheter removed. Small resections of the right frontal lobe were performed in large dogs. Magnetic resonance (MR) images were obtained and used to assess tissue response and to measure the conformance between the resection cavity wall and the balloon surface. In four animals a dose ranging from 36 to 59 Gy was delivered. Neurological status and histological characteristics of the brain were assessed in all dogs. Implantation and explantation as well as inflation and deflation of the device were easily accomplished and well tolerated. The device was easily visualized on MR images, which demonstrated the expected postsurgical changes. The resection cavity and the balloon were highly conformal (range 93-100%). Histological changes to the cavity margin were consistent with those associated with surgical trauma. Additionally, radiation-related changes were observed at the margins of the resection cavity in dogs in which the brain was irradiated. CONCLUSIONS: This balloon catheter and 125I radiotherapy solution system can safely and reliably deliver radiation to the margins of brain cavities created by tumor resection. Results of this study showed that intracranial pressure changes due to balloon inflation and deflation were unremarkable and characteristic of the imaging properties and radiation safety profile of the device prior to its clinical evaluation. Clinically relevant brachytherapy (adequate target volume and total dose) was accomplished in all four animals subjected to treatment.