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Induction of DNA DSBs by low-LET radiations reflects clustered damage produced predominantly by low-energy, secondary electron "track ends". Cell inactivation and induction of DSBs and their rejoining, assayed using pulsed-field gel electrophoresis, were determined in Chinese hamster V79-4 cells irradiated as a monolayer with characteristic carbon K-shell (CK) (0.28 keV), aluminum K-shell (AlK) (1.49 keV), and titanium K-shell (TiK) (4.55 keV) ultrasoft X rays under aerobic and anaerobic conditions. Relative to (60)Co gamma rays, the relative biological effectiveness (RBE) for cell inactivation at 10% survival and for induction of DSBs increases as the photon energy of the ultrasoft X rays decreases. The RBE values for cell inactivation and for induction of DSBs by CK ultrasoft X rays are 2.8 +/- 0.3 and 2.7 +/- 0.3, respectively, and by TiK ultrasoft X rays are 1.5 +/- 0.1 and 1.4 +/- 0.1, respectively. Oxygen enhancement ratios (OERs) of approximately 2 for cell inactivation and induction of DSBs by ultrasoft X rays are independent of the photon energy. The time scale for rejoining of DNA DSBs is similar for both ultrasoft X rays and 60Co gamma rays. From the size distribution of small DNA fragments down to 0.48 kbp, we concluded that DSBs are induced randomly by CK and AlK ultrasoft X rays. Therefore, ultrasoft X rays are more efficient per unit dose than gamma radiation at inducing DNA DSBs, the yield of which increases with decreasing photon energy.

Background Perioperative hypothermia has been reported to increase the occurrence of cardiovascular complications. By increasing the activity of sympathetic nervous system, perioperative hypothermia also has the potential to increase cardiac injury and dysfunction associated with subarachnoid hemorrhage. Methods The Intraoperative Hypothermia for Aneurysm Surgery Trial randomized patients undergoing cerebral aneurysm surgery to intraoperative hypothermia (n = 499, 33.3 degrees +/- 0.8 degrees C) or normothermia (n = 501, 36.7 degrees +/- 0.5 degrees C). Cardiovascular events (hypotension, arrhythmias, vasopressor use, myocardial infarction, and others) were prospectively followed until 3-month follow-up and were compared in hypothermic and normothermic patients. A subset of 62 patients (hypothermia, n = 33; normothermia, n = 29) also had preoperative and postoperative (within 24 h) measurement of cardiac troponin-I and echocardiography to explore the association between perioperative hypothermia and subarachnoid hemorrhage-associated myocardial injury and left ventricular function. Results There was no difference between hypothermic and normothermic patients in the occurrence of any single cardiovascular event or in composite cardiovascular events. There was no difference in mortality (6%) between groups, and there was only a single primary cardiovascular death (normothermia). There was no difference between hypothermic and normothermic patients in postoperative versus preoperative left ventricular regional wall motion or ejection fraction. Compared with preoperative values, hypothermic patients had no postoperative increase in cardiac troponin-I (median change 0.00 microg/l), whereas normothermic patients had a small postoperative increase (median change + 0.01 microg/l, P = 0.038). Conclusion In patients undergoing cerebral aneurysm surgery, perioperative hypothermia was not associated with an increased occurrence of cardiovascular events.

Background Although hypothermia and barbiturates improve neurologic outcomes in animal temporary focal ischemia models, the clinical efficacy of these interventions during temporary occlusion of the cerebral vasculature during intracranial aneurysm surgery (temporary clipping) is not established. Methods A post hoc analysis of patients from the Intraoperative Hypothermia for Aneurysm Surgery Trial who underwent temporary clipping was performed. Univariate and multivariate logistic regression methods were used to test for associations between hypothermia, supplemental protective drug, and short- (24-h) and long-term (3-month) neurologic outcomes. An odds ratio more than 1 denotes better outcome. Results Patients undergoing temporary clipping (n = 441) were assigned to intraoperative hypothermia (33.3 degrees +/- 0.8 degrees C, n = 208) or normothermia (36.7 degrees +/- 0.5 degrees C, n = 233), with 178 patients also receiving supplemental protective drug (thiopental or etomidate) during temporary clipping. Three months after surgery, 278 patients (63%) had good outcome (Glasgow Outcome Score = 1). Neither hypothermia (P = 0.847; odds ratio = 1.043, 95% CI = 0.678-1.606) nor supplemental protective drug (P = 0.835; odds ratio = 1.048, 95% CI = 0.674-1.631) were associated with 3-month Glasgow Outcome Score. The effect of supplemental protective drug did not significantly vary with temperature. The effects of hypothermia and protective drug did not significantly vary with temporary clip duration. Similar findings were made for 24-h neurologic status and 3-month Neuropsychological Composite Score. Conclusion In the Intraoperative Hypothermia for Aneurysm Surgery Trial, neither systemic hypothermia nor supplemental protective drug affected short- or long-term neurologic outcomes of patients undergoing temporary clipping.

Dendritic cells (DC) are professional APC that control the balance between T cell immunity and tolerance. Genetic engineering of DC to regulate the outcome of the immune response is an area of intense research. Galectin (gal)-1 is an endogenous lectin that binds to glycoproteins and exerts potent regulatory effects on T cells. Consequently, gal-1 participates in central deletion of thymocytes and exerts therapeutic effects on experimental models of T cell-mediated autoimmune disorders and graft-vs-host disease. Together, these observations strongly indicate that engineering DC to express transgenic (tg) gal-1 may be beneficial to treat T cell-mediated disorders. In this study, we have investigated the impact of the expression of high levels of tg gal-1 on maturation/activation of DC and on their T cell stimulatory function. Murine DC were transduced with a recombinant adenovirus encoding hu gal-1 (gal-1-DC). Tg gal-1 was exported by a nonclassical pathway through exosomes and was retained on the DC surface inducing segregation of its ligand CD43. Expression of tg gal-1 triggered activation of DC determined by induction of a more mature phenotype, increased levels of mRNA for proinflammatory cytokines, and enhanced ability to stimulate naive T cells. Conversely, gal-1-DC induced rapid apoptosis of activated T cells. In vivo, gal-1-DC increased significantly the sensitization phase of contact hypersensitivity assays while inducing a drastic inhibition of the elicitation phase by triggering apoptosis of activated T cells in the dermis. Gal-1-DC represent a novel tool to control differentially the afferent and efferent arms of the T cell response.

Diabetes is a disease of epidemic proportions and is on the rise worldwide. Gene therapy has been actively pursued but limited by technical hurdles and profound inefficiency of direct gene transfer to the pancreas in vivo. Here, we show that, for the first time, appropriate serotypes of adeno-associated virus (AAV), coupled with a double-stranded vector DNA cassette, enable extensive and long-term in vivo gene transfer in the adult mouse pancreas by three different delivery methods. Intraperitoneal and intravenous delivery of AAV8 effectively transduced exocrine acinar cells as well as endocrine β-cells, while local pancreatic intraductal delivery of AAV6 showed the best efficiency in the β-cells among all AAV serotypes tested in this study. Nearly the entire islet population showed gene transfer but with distinct gene transfer efficiency and patterns when different delivery methods and vectors were used. Importantly, localized gene delivery coupled with an insulin promoter allowed extensive yet specific gene expression in the β-cells. These effective new methods should provide useful tools to study diabetes pathogenesis and gene therapy.

We have created a novel cellular vehicle for gene therapy of malignant gliomas by transfection of murine bone marrow stroma cells (MSCs) with a cDNA encoding epidermal growth factor receptor (EGFR). These cells (EGFR-MSCs) demonstrate enhanced migratory responses toward glioma-conditioned media in comparison to primary MSCs in vitro. Enhanced migration of EGFR-MSC was at least partially dependent on EGF-EGFR, PI3-, MAP kinase kinase, and MAP kinases, protein kinase C, and actin polymerization. Unlike primary MSCs, EGFR-MSCs were resistant to FasL-mediated cytotoxicity and were capable of stimulating allogeneic mixed lymphocyte reaction, suggesting EGFR-MSCs possess suitable characteristics as vehicles for brain tumor immuno-gene therapy. Following injection at various sites, including the contralateral hemisphere in the brain of syngeneic mice, EGFR-MSCs were able to migrate toward GL261 gliomas or B16 melanoma in vivo. Finally, intratumoral injection with EGFR-MSC adenovirally engineered to secrete interferon-alpha to intracranial GL261 resulted in significantly prolonged survival in comparison to controls. These data indicate that EGFR-MSCs may serve as attractive vehicles for infiltrating brain malignancies such as malignant gliomas.

Exosomes are nanovesicles released by leukocytes and epithelial cells. Although their function remains enigmatic, exosomes are a source of antigen and transfer functional major histocompatibility complex (MHC)–I/peptide complexes to dendritic cells (DCs) for CD8+ T-cell activation. Here we demonstrate that exosomes also are internalized and processed by immature DCs for presentation to CD4+ T cells. Endocytosed exosomes are sorted into the endocytic compartment of DCs for processing, followed by loading of exosome-derived peptides in MHC-II molecules for presentation to CD4+ T cells. Targeting of exosomes to DCs is mediated via milk fat globule (MFG)–E8/lactadherin, CD11a, CD54, phosphatidylserine, and the tetraspanins CD9 and CD81 on the exosome and αv/β3 integrin, and CD11a and CD54 on the DCs. Circulating exosomes are internalized by DCs and specialized phagocytes of the spleen and by hepatic Kupffer cells. Internalization of blood-borne allogeneic exosomes by splenic DCs does not affect DC maturation and is followed by loading of the exosome-derived allopeptide IEα52-68 in IAb by host CD8α+ DCs for presentation to CD4+ T cells. These data imply that exosomes present in circulation or extracellular fluids constitute an alternative source of self- or allopeptides for DCs during maintenance of peripheral tolerance or initiation of the indirect pathway of allorecognition in transplantation.

Effective presentation of tumor antigens by dendritic cells (DCs) is considered to be essential for the induction of antitumor T-cell responses. Apoptotic and necrotic tumors have been noted to be a robust antigen source for DCs. Because glioma cells undergo apoptosis after transfection with the type I interferon (IFN) gene and type I IFNs promote the stimulatory activity of DCs, we hypothesized that transfection of glioma cells with type I IFN genes and provision of DCs would promote particularly effective antitumor activity by both facilitating apoptosis of glioma cells and the presentation of the glioma antigens, thereby inducing specific immune responses against glioma cells. We have previously reported the proof of this hypothesis in vitro and in a subcutaneous tumor model. Here we report an extension of this approach in intracranial (i.c.) gliomas using adenoviral IFN-alpha (Ad-IFN-alpha) vector. Mice bearing day-5 i.c. GL261 glioma received sequential intratumoral (i.t.) delivery of Ad-IFN-alpha and bone marrow-derived syngeneic DCs. This treatment prolonged survival in that nine of 17 animals survived long term (> 60 days versus 0 of 10 control animals). Specific CTL activity was demonstrated following this regimen in the cervical lymph nodes, and the therapeutic efficacy was dependent upon CD8+ cells. Furthermore, these animals were protected against subsequent re-challenge with GL261 gliomas. DCs injected i.t. survived in the tumor and migrated into cervical lymph node. In vitro migration assays revealed the ability of DCs to migrate toward the tumor, suggesting that i.t. injected DCs migrate through the glioma. Taken together, this combination of gene therapy and cellular immunotherapy may be an effective future strategy for treating human gliomas.

We demonstrate two novel approaches to enhance interactions of polymer-immobilized biomolecules with their substrates. In the first approach, diisopropylfluorophosphatase (DFPase) containing poly(urethane) (PU) coatings were made microporous by incorporating, then extracting, poly(ethylene glycol)-based diesters as porogens. Incorporation of 2% w/w porogen increased the effective diffusion coefficient of diisopropylfluorophosphate (DFP) through the coatings by 30% and increased the apparent turnover number of immobilized DFPase 3-fold. In the second approach, prior to immobilization, hydrophobic modification of DFPase was achieved through its conjugation with a dimer/trimer mixture of a uretdione based on 1,6-diisocyanatohexane. When the hydrophobically modified DFPase was immobilized in coatings, catalytic activity was 4-fold higher than that of the equivalent, immobilized, native DFPase. This activity enhancement was independent of the presence or absence of pores. Confocal microscopy images of coatings containing fluorescently labeled lysozyme show that the native enzyme is distributed uniformly over the entire thickness of the coatings. Hydrophobically modified and fluorescently labeled lysozyme is accumulated only in the upper 10 μm cross-sectional layer of a 100 μm-thick coating. Interactions of bioplastics with their substrates are tunable either by pore induction in a polymer or by directed migration of the hydrophobically modified biomolecule to the desired location. The latter approach has broad implications, including overcoming mass transfer limitations experienced by immobilized biocatalysts. © 2004 Wiley Periodicals, Inc.

The role of the NH(2)-terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH(2)-terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and whole-cell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 +/- 1.3 min compared with 3.2 +/- 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH(2) terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27 degrees C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH(2) terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.

Islet transplantation therapy would be applicable to a wider range of diabetic patients if donor islet acceptance and protection were possible without systemic immunosuppression of the recipient. To this aim, gene transfer to isolated donor islets ex vivo is one method that has shown promise. This study examines the combined effect of selected immunomodulatory and anti-inflammatory genes known to extend the functional viability of pancreatic islet grafts in an autoimmune system. These genes, indoleamine 2,3-dioxygenase (IDO), manganese superoxide dismutase (MnSOD), and interleukin (IL)-1 receptor antagonist protein (IRAP), were transferred to isolated NOD donor islets ex vivo then transplanted to NODscidrecipients and evaluated in vivo after diabetogenic T-cell challenge. The length of time the recipient remained euglycemic was used to measure the ability of the transgenes to protect the graft from autoimmune destruction. Although the results of these cotransfections gave little evidence of a synergistic relationship, they were useful to show that gene combinations can be used to more efficiently protect islet grafts from diabetogenic T cells.

Our previous studies demonstrated that adoptive transfer of dendritic cells (DC) prevents diabetes in young nonobese diabetic (NOD) mice by inducing regulatory T(H)2 cells. In this report, as a means of treating NOD mice with more advanced insulitis, we infected DC with adenoviral vectors expressing interleukin (IL)-4 (Ad.IL-4), eGFP (Ad.eGFP), or empty vector (Ad psi 5). DC infected with any of the Ad vectors expressed higher levels of CD40, CD80, and CD86 molecules than uninfected DC and Ad.IL-4 DC produced IL-4 after lipopolysaccharide (LPS) and interferon (IFN)-gamma stimulation. Ad-infected DC efficiently stimulated allogeneic T cells, and cultures of T cells with Ad.IL-4 DC produced lower levels of IFN-gamma and marginally higher levels of IL-4. In vivo studies demonstrated that the Ad.eGFP DC trafficked to the pancreatic lymph nodes within 24 hr of intravenous administration, and could be visualized in the T cell areas of the spleen. The intrapancreatic IFN-gamma:IL-4 or IFN-gamma:IL-10 cytokine ratios were lower in 10-week-old mice treated with Ad.IL-4 DC, and these mice were significantly protected from disease. These results demonstrate, for the first time, that genetically modified DC can prevent diabetes in the context of advanced insulitis.

We combined confocal and live-cell imaging with a novel molecular strategy aimed at revealing mechanisms underlying glucose-regulated insulin vesicle secretion. The 'Ins-C-GFP' reporter monitors secretory peptide targeting, trafficking, and exocytosis without directly tagging the mature secreted peptide. We trapped a green fluorescent protein (GFP) reporter in equimolar quantity within the secretory vesicle by fusing it within the C peptide of proinsulin which only after nascent vesicle sealing and acidification is cleaved from the mature secreted A and B chains of insulin. Ins-C-GFP expression in mouse islets without fail exhibited punctate distribution of green fluorescence by confocal microscopy. Ins-C-GFP colocalized GFP with insulin at vesicle dense cores by immuno-electron microscopy. Glucose stimulation decreased vesicle fluorescence coordinately with enhanced secretion from islets of C-GFP detected by anti-GFP Western blots, and of insulin detected by anti-insulin radioimmunoassay. An insulin secretagogue with a red fluorescent label, glibenclamide BODIPY TR, was applied to islets expressing Ins-C-GFP. The stimulus response was imaged as a rise in red secretagogue leading to marked loss in green granules. Since neuropeptides as well as peptide hormones are processed from propeptides after sealing of secretory granules, vesicle trapping likely is widely applicable for studies on targeting, trafficking, and regulated release of secretory peptides.

To determine whether there is a relationship between the genetics underlying the susceptibility to radiation-induced leukaemia in CBA/H (acute myeloid leukaemia, AML) and C57BL/6 (thymic lymphoma, TL) mice, and the genetics underlying the sensitivity of CBA/H (sensitive) and C57BL/6 (resistant) mice to radiation-induced chromosomal instability.CBA/H, (CBA/H x C57BL/6)F1, F1 x CBA/H, F1 x C57BL/6 and F1 x F1 mice were exposed to a single acute dose of 3.0 Gy X-rays. AML and TL were diagnosed over the subsequent 30 months.There was no statistically significant difference in the incidence of AML in F1, F1 x F1, F1 x CBA/H and F1 x C57BL/6 mice, which was approximately 50% that in CBA/H mice. AML susceptibility is therefore a dominant polygenic trait, and both susceptibility and resistance (variable penetrance) CBA/H and C57BL/6 loci are involved. The incidence of TL in the FM and F1 x CBA/H mice was negligible, indicating that TL susceptibility is a recessive trait. As the TL incidence in the F1 x C57BL/6 mice was about half that in C57BL/6 mice, one recessive locus is probably involved.AML susceptibility in CBA/H mice is a dominant trait in contrast to the recessive inheritance of CBA/H sensitivity to radiation-induced chromosomal instability. TL-susceptibility in C57BL/6 is a recessive trait in contrast to the dominant inheritance of C57BL/6 resistance to radiation-induced chromosomal instability.

Burns (3 mm in diameter, 50°C, 20 s duration) were induced on the skin of anaesthetised hairless mice. Anaesthesia was maintained throughout all experiments. Subsurface changes in the microvasculature at the burn site were imaged confocally following i.v. injection of fluorescently labelled (FITC) dextran. Blood cells moving through dermal blood vessels were seen and recorded on video tape. Multiple adjacent 2-D confocal images of the burn site and surrounding areas were assembled and enabled microscopic vascular imaging of the whole burn area (including zones of coagulation, stasis and hyperaemia) and the surrounding normal vessels. This mapping of the burn area by fibre optic confocal imaging (FOCI) in vivo demonstrated good congruence with vascular casts (Microfil MV-120, Flow tech, USA) made at 4 h post burn. This study demonstrates the usefulness of FOCI for in vivo vascular imaging in burns.

Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample phi ssh = [1.5 +/- 0.1] x 10(4), argon-saturated sample phi ssh = [0.9 +/- 0.1] x 10(4). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand break-age (air-saturated sample phi fpg = [33.1 +/- 3.1] x 10(-4), argon-saturated sample phi fpg = [23.8 +/- 2.6] x 10(-4). This result indicates that 193 nm light induces other modification(s) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.

OBJECTIVE: We examined the incidence of perioperative fever and its relationship to outcome among patients enrolled in the Intraoperative Hypothermia for Aneurysm Surgery Trial. METHODS: One thousand patients with initial World Federation of Neurological Surgeons grades of I to III undergoing clipping of intracranial aneurysms after subarachnoid hemorrhage were randomized to intraoperative normothermia (36 degrees C-37 degrees C) or hypothermia (32.5 degrees C-33.5 degrees C). Fever (> or =38.5 degrees C) and other complications (including infections) occurring between admission and discharge (or death) were recorded. Functional and neuropsychologic outcomes were assessed 3 months postoperatively. The primary outcome variable for the trial was dichotomized Glasgow Outcome Scale (good outcome versus all others). RESULTS: Fever was reported in 41% of patients. In 97% of these, fever occurred in the postoperative period. The median time from surgery to first fever was 3 days. All measures of outcome were worse in patients who developed fever, even in those without infections or who were World Federation of Neurological Surgeons grade I. Logistic regression analyses were performed to adjust for differences in preoperative factors (e.g., age, Fisher grade, initial neurological status). This demonstrated that fever continued to be significantly associated with most outcome measures, even when infection was added to the model. An alternative stepwise model selection process including all fever-related measures from the preoperative and intraoperative period (e.g., hydrocephalus, duration of surgery, intraoperative blood loss) resulted in the loss of significance for dichotomized Glasgow Outcome Scale, but significant associations between fever and several other outcome measures remained. After adding postoperative delayed ischemic neurological deficits to the model, only worsened National Institutes of Health Stroke Scale score, Barthel Activities of Daily Living index, and discharge destination (home versus other) remained independently associated with fever. CONCLUSION: These findings suggest that fever is associated with worsened outcome in surgical subarachnoid hemorrhage patients, although, because the association between fever and the primary outcome measure for the trial is dependent on the covariates used in the analysis (particularly operative events and delayed ischemic neurological deficits), we cannot rule out the possibility that fever is a marker for other events. Only a formal trial of fever treatment or prevention can address this issue.

Exosomes are nanovesicles released by different cell types including dendritic cells (DCs). The fact that exosomes express surface MHC-peptide complexes suggests that they could function as Ag-presenting vesicles or as vehicles to spread allogeneic Ags for priming of anti-donor T cells during elicitation of graft rejection or induction/maintenance of transplant tolerance. We demonstrate that circulating exosomes transporting alloantigens are captured by splenic DCs of different lineages. Internalization of host-derived exosomes transporting allopeptides by splenic DCs leads to activation of anti-donor CD4 T cells by the indirect pathway of allorecognition, a phenomenon that requires DC-derived, instead of exosome-derived, MHC class II molecules. By contrast, allogeneic exosomes are unable to stimulate direct-pathway T cells in vivo. We demonstrate in mice that although graft-infiltrating leukocytes release exosomes ex vivo, they do not secrete enough concentrations of exosomes into circulation to stimulate donor-reactive T cells in secondary lymphoid organs. Instead, our findings indicate that migrating DCs (generated in vitro or isolated from allografts), once they home in the spleen, they transfer exosomes expressing the reporter marker GFP to spleen-resident DCs. Our results suggest that exchange of exosomes between DCs in lymphoid organs might constitute a potential mechanism by which passenger leukocytes transfer alloantigens to recipient’s APCs and amplify generation of donor-reactive T cells following transplantation.

The interaction of donor passenger leukocytes and host leukocytes in recipient secondary lymphoid tissues during the early posttransplantation period is crucial in directing host immune reactions toward allograft rejection or acceptance. Responsible T cell clones could be activated through the direct and indirect pathways of allorecognition. We examined the role of the indirect pathway in liver transplantation (LT) tolerance by depleting host antigen-presenting cells (APC) with phagocytic activity [e.g., cluster domain (CD)68+/CD163+ macrophages, CD11c+ dendritic cells (DC)] using liposome-encapsulating clodronate (LP-CL). After Lewis rat cell or liver graft transplantation, Brown Norway (BN) rat recipients pretreated with LP-CL showed a significantly reduced type 1 helper T cell cytokine up-regulation than control-LP-treated recipients. In the LT model, LP-CL treatment and host APC depletion abrogated hepatic tolerance; Lewis liver grafts in LP-CL-treated-BN recipients developed mild allograft rejection, failed to maintain donor major histocompatibility complex (MHC) class II+ leukocytes, and developed chronic rejection in challenged donor heart allografts, while control-LP-treated BN recipients maintained tolerance status and donor MHC class II+ hepatic leukocytes. Furthermore, in the BN to Lewis LT model, LP-CL recipient treatment abrogated spontaneous hepatic allograft acceptance, and graft survival rate was reduced to 43% from 100% in the control-LP group. In conclusion, the study suggests that host cells with phagocytic activity could play significant roles in developing LT tolerance.

Metastatic seeding leads to most of the morbidity from carcinomas. However, little is known of this key event as current methods to study the cellular behaviors utilize nonrepresentative in vitro models or follow indirect subsequent developments in vivo. Therefore, we developed a system to visualize over a multiday to multiweek period the interactions between tumor cells and target organ parenchyma. We employ an ex vivo microscale perfusion culture system that provides a tissue-relevant environment to assess metastatic seeding behavior. The bioreactor recreates many features of the fluid flow, scale, and biological functionality of a hepatic parenchyma, a common site of metastatic spread for a wide range of carcinomas. As a test of this model, prostate and breast carcinoma cells were introduced. Tumor cell invasion and expansion could be observed by two-photon microscopy of red fluorescent protein (RFP)- and CellTracker-labeled carcinoma cells against a green fluorescent protein (GFP)-labeled hepatic tissue bed over a 14-day period. Tumors visible to the naked eye could be formed by day 25, without evident necrosis in the >0.3-mm tumor mass. These tumor cells failed to grow in the absence of the supporting three-dimensional (3D) hepatic microtissue, suggesting paracrine or stromal support function for the liver structure in tumor progression. Initial ultrastructural studies suggest that early during the tumor-parenchyma interactions, there are extensive interactions between and accommodations of the cancer and host cells, suggesting that the tumor-related epithelial-mesenchymal transition (EMT) reverts, at least transiently, to promote metastatic seeding. In sum, our 3D ex vivo organotypic liver tissue system presents a critical vehicle to examine tumor-host interactions during cancer metastasis and/or invasion. It also circumvents current limitations in assays to assess early events in metastasis, and provides new approaches to study molecular events during tumor progression.

The effects of inhalation of radon/radon decay products at different total doses, dose rates and 'unattached' fractions were investigated in a life span study in rats.1574 rats inhaled radon/radon decay products in a purpose-built recirculating exposure system that provided stable/reproducible exposure conditions. 501 were maintained as controls.Lung tumour incidences were significantly elevated in most exposed groups. The study power was insufficient to resolve the shape of the dose and dose rate response curves, but combination of this data with that from other studies demonstrated that for high cumulative exposures, the lifetime excess absolute risk increases with increasing exposure durations and for low cumulative exposures the opposite trend occurs. Exposure did not increase leukaemia incidences. A small number of non-lung tumour types including mammary fibroadenoma showed elevated incidences in some exposed groups, however not consistently across all exposure groups and showed no dose or dose rate relationship.Radon/radon decay product exposure caused excess lung tumours in rats along with limited non-lung effects. The results are consistent with the findings that at low cumulative exposures decreasing exposure concentrations or protracting the time over which the dose is delivered, reduces lung tumour risk. At higher levels, decreasing exposure concentrations or protracting exposure time increases lung tumour risk.

The tensile strength and stiffness of load-bearing soft tissues are dominated by their collagen fiber orientation. While microgrooved substrates have demonstrated a capacity to orient cells and collagen in monolayer tissue culture, tissue engineering (TE) scaffolds are structurally distinct in that they consist of a three-dimensional (3-D) open pore network. It is thus unclear how the geometry of these open pores might influence cell and collagen orientation. In the current study we developed an in vitro model system for quantifying the capacity of large scale (∼200 μm), geometrically well-defined open pores to guide cell and collagen orientation in engineered tissues. Non-degradable scaffolds exhibiting a grid of 200 μm wide rectangular pores (1:1, 2:1, 5:1, and 10:1 aspect ratios) were fabricated from a transparent epoxy resin via high-resolution stereolithography. The scaffolds ( n = 6 per aspect ratio) were surface modified to support cell adhesion by covalently grafting GRGDS peptides, sterilized, and seeded with neonatal rat skin fibroblasts. Following 4 weeks of static incubation, the resultant collagen orientation was assessed quantitatively by small angle light scattering (SALS), and cell orientation was evaluated by laser confocal and scanning electron microscopy. Cells adhered to the struts of the pores and proceeded to span the pores in a generally circumferential pattern. While the cell and collagen orientations within 1:1 aspect ratio pores were effectively random, higher aspect ratio rectangular pores exhibited a significant capacity to guide global cell and collagen orientation. Preferential alignment parallel to the long strut axis and decreased spatial variability were observed to occur with increasing pore aspect ratio. Intra-pore variability depended in part on the spatial uniformity of cell attachment around the perimeter of each pore achieved during seeding. Evaluation of diamond-shaped pores [Sacks, M.S. et al., 1997. J. Biomech. Eng. 119(1), 124–127] suggests that they are less sensitive to initial conditions of cell attachment than rectangular pores, and thus more effective in guiding engineered tissue cell and collagen orientation.

Islet transplantation is a promising cure for diabetes. However, inflammation, allorejection, and recurrent autoimmune damage all may contribute to early graft loss. Pancreatic islets express lower levels of antioxidant genes than most other tissues of the body, and β-cells in particular are sensitive to oxidative damage. Therefore, damage from oxidative stress may pose a major obstacle to islet replacement therapy in that both the islet isolation and transplantation processes generate oxygen radicals. To determine whether antioxidant gene overexpression in isolated pancreatic islets can prevent oxidative damage and prolong islet function after transplantation, we used the NOD mouse model to study oxidative stress encountered during both transplantation and autoimmune attack. We transferred an antioxidant gene, manganese superoxide dismutase (MnSOD), by adenoviral infection into isolated islets that were transplanted into streptozotocin-treated NODscid recipient mice. Functioning islet grafts were subsequently exposed to diabetogenic spleen cells and monitored until graft failure. The results show that islet grafts overexpressing MnSOD functioned ∼50% longer than control grafts. This significant prolongation of graft function suggests that the antioxidant activity of MnSOD is beneficial to transplanted islet survival and may be used in combination with other strategies aimed at islet graft protection.

Urethanes are frequently used in biomedical applications because of their excellent biocompatibility. However, their use has been limited to bioresistant polyurethanes. The aim of this study was to develop a nontoxic biodegradable polyurethane and to test its potential for tissue compatibility. A matrix was synthesized with pentane diisocyanate (PDI) as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix. The matrix was biodegradable in an aqueous solution at 37°C in vitro as well as in vivo. The polymer was mechanically stable at body temperatures and exhibited a glass transition temperature (Tg) of 67°C. The porosity of the polymer network was between 10 and 2000 μm, with the majority of pores between 100 and 300 μm in diameter. This porosity was found to be adequate to support the adherence and proliferation of bone-marrow stromal cells (BMSC) and chondrocytes in vitro. The degradation products of the polymer were nontoxic to cells in vitro. Subdermal implants of the PDI–sucrose matrix did not exhibit toxicity in vivo and did not induce an acute inflammatory response in the host. However, some foreign-body giant cells did accumulate around the polymer and in its pores, suggesting its degradation is facilitated by hydrolysis as well as by giant cells. More important, subdermal implants of the polymer allowed marked infiltration of vascular and connective tissue, suggesting the free flow of fluids and nutrients in the implants. Because of the flexibility of the mechanical strength that can be obtained in urethanes and because of the ease with which a porous microtexture can be achieved, this matrix may be useful in many tissue-engineering applications. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 64A: 242–248, 2003

We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of non-functional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted into the extracellular loop between transmembrane domains S3 and S4. Surface expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp techniques. Although deletion of the last 14 amino acids of hIK1 (L414STOP) had no effect on plasma membrane expression and function, deletion of the last 26 amino acids (K402STOP) resulted in a complete loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu(406) (L399A/L406A) completely abrogated membrane localization. Additional mutations within the heptad repeat (L385A/L392A, L392A/L406A) or of the a positions (I396A/L403A) resulted in a near-complete loss of membrane-localized channel. In contrast, mutating individual leucines did not compromise channel trafficking or function. Both membrane localization and function of L399A/L406A could be partially restored by incubation at 27 degrees C. Co-immunoprecipitation studies demonstrated that leucine zipper mutations do not compromise multimer formation. In contrast, we demonstrated that the leucine zipper region of hIK1 is capable of co-assembly and that this is dependent upon an intact leucine zipper. Finally, this leucine zipper is conserved in another member of the gene family, SK3. However, mutation of the leucine zipper in SK3 had no effect on plasma membrane localization or function. In conclusion, we demonstrate that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.

Under steady-state conditions, internalization of self-antigens embodied in apoptotic cells by dendritic cells (DCs) resident in peripheral tissue followed by DC migration and presentation of self-peptides to T cells in secondary lymphoid organs are key steps for induction and maintenance of peripheral T-cell tolerance. We show here that, besides this traffic of apoptotic cells mediated by peripheral tissue-resident DCs, splenic marginal zone DCs rapidly ingest circulating apoptotic leukocytes, process apoptotic cell-derived peptides into major histocompatibility complex class II (MHC-II) molecules, and acquire CD8alpha during their mobilization to T-cell areas of splenic follicles. Because apoptotic cells activate complement and some complement factors are opsonins for phagocytosis and play roles in the maintenance of peripheral tolerance, we investigated the role of complement receptors (CRs) in relation to phagocytosis of apoptotic cells by DCs. Apoptotic cell uptake by marginal zone DCs was mediated in part via CR3 (CD11b/CD18) and, to a lesser extent, CR4 (CD11c/CD18) and was reduced significantly in vivo in hypocomplementemic animals. Following phagocytosis of apoptotic cells, DCs exhibited decreased levels of mRNA and secretion of the proinflammatory cytokines interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-12p70, and tumor necrosis factor alpha (TNF-alpha), without effect on the anti-inflammatory mediator transforming growth factor beta1 (TGF-beta1). This selective inhibitory effect was at least partially mediated through C3bi-CD11b/CD18 interaction. Characterization of apoptotic cell/DC interaction and its outcome provides insight into the mechanisms by which apoptotic cells affect DC function without disrupting peripheral tolerance.

The absence of effective conventional therapy for most cancer patients justifies the application of novel, experimental approaches. One alternative to conventional cytotoxic agents is a more defined molecular approach for cancer immune treatment; promotion of the immune system specifically to target and eliminate tumor cells on the basis of expression of tumor-associated antigens (TAA). TAA could be presented to T-cells by professional antigen-presenting cells (APC) that generate a more efficient and effective anti-tumor immune response. In fact, it has been well documented that dendritic cells, the most immunologically potent APC, are capable of recognizing, processing and presenting TAA, in turn initiating a specific antitumor immune response. Results from several laboratories and clinical trials suggested significant but still limited efficacy of TAA-pulsed dendritic cells administered to tumor-bearing hosts. Following such delivery, it is fundamentally necessary to dynamically assess cell abundance within the microenvironment of the tumor in the presence of the appropriate therapeutic agent. Multiphoton microscopy was used to assess the trafficking of pulsed dendritic cells and other APC in skin, lymph nodes and brain of several animal tumor models, following different routes of administration.© (2002) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Dendritic cells (DCs) can be identified according to many different phenotypic characteristics, related both to their tissue of origin and stage of differentiation. Different populations can be fluorescently immunolabeled using antibodies to one or more molecules expressed by the cells. Fluorescence microscopy can be used to examine the specific locations of different populations of endogenous DCs within sections of various tissues, tonsil, and their interactions with other types of cells within the tissue. Electron microscopy (EM) is useful for freshly sorted or isolated dendritic cells or cultured dendritic cells. DCs have unique ultrastructural characteristics that set them apart from other mesenchymal cells, including leukocytes such as monocytes, T, and B cells. DCs are often best identified from other leukocytes using surface topology afforded by scanning electron microscopy (SEM). This technique allows the visualization of the membrane “veils” or “dendrites,” which readily distinguish DCs from other cell types.

A fluorescence confocal microscopy technique was employed to obtain subsurface images of nerve and microvascular structure in the vas deferens and colon of the living rat. The use of dual labelling with vital dyes and 2-channel confocal acquisition allowed differentiation of microscopic structure at both low and higher magnification. Characteristic staining patterns of nerves and blood vessels were repeatedly obtained in each tissue, suggesting the potential of this technique for studying morphological changes associated with surgical procedures and/or models of neuronal or vascular pathology.

Burn injury causes vascular thrombosis and occlusion by thermal damage to the vascular network in the dermis. In this study, fibre optic confocal imaging (FOCI) and laser doppler flowmetry were used to detect changes in vascular morphology and local dermal blood flux over 4 h, in three defined zones after a thermal burn (50°C, 20s duration, 3 mm in diameter) was induced on fully anaesthetised hairless mice. FITC-dextran (i.v.) was used to enable FOCI of vascular morphology including three-dimensional imaging of the burn site and its surrounding areas. Samples of the affected areas were collected for conventional histology, including Masson's trichrome. There was vascular damage in the zone of coagulation which showed no change during the 4 h period. The zone of stasis showed an initial reduction in blood flux and confocal imaging of the area indicated significant vessel leakage during the first 2 h which later improved. The zone of hyperaemia showed an initial increase in total blood flux and confocal imaging of the area showed initial blood vessel dilatation. This study demonstrates that FOCI is a useful non-invasive tool in the assessment of vascular changes in thermal burns in vivo, and compares the findings of FOCI with those from laser doppler flowmetry and histology.

The Gorlin syndrome, or naevoid basal-cell carcinoma syndrome (NBCS) is an autosomal dominant cancer prone disease (at risk of multiple basal cell carcinomas, and other malignant or benign proliferations). We have previously reported data from peripheral blood lymphocytes of patients with this condition, showing a significant level of spontaneous chromatid and chromosome rearrangements and an overall lengthening of the cell cycle. In this paper, we confirm this disease to be a chromosome instability syndrome from studies on fibroblasts of 5 patients. Spontaneous chromosomal rearrangements, an increased frequency of sister chromatid exchanges and a slowing of the cell cycle were found, compared to age-matched control material. There was also an increased sensitivity to aberration production by mechlorethamine in patient fibroblasts. The chromosome instability we found was not restricted to a given cell lineage, but appears to be part of the general condition of this syndrome. The recently discovered gene responsible for Gorlin syndrome, PTC (or PTCH), encodes a transmembrane protein with yet poorly known functions. However, the demonstration of Gorlin syndrome as a chromosome instability syndrome suggests that this protein has a role in DNA maintenance, repair and/or replication.

Menkes disease (MD) is caused by a defect in copper homeostasis and has a recognised mouse model, mottled (Atp7aMo). Copper uptake and retention assays performed on fibroblast cultures have been used successfully for pre- and postnatal diagnosis of Menkes disease. We report here the results of these assays applied to primary fibroblast cultures established from nine independent mottled alleles associated with phenotypes of varying severity maintained on identical genetic backgrounds. No significant differences were found between the different alleles, or between the mottled cultures and fibroblasts established from MD patients. Thus, in the mouse, the data obtained for copper retention/uptake at the cellular level do not correlate with the severity of the phenotype.

Acute myeloid leukaemias induced by ionizing radiation in mouse are characterized by chromosome (chr) 2 aberrations. While it is known that chr 2 aberrations form early and in abundance post-irradiation, unequivocal evidence for hypersensitivity of chr 2 in the first post-irradiation mitoses is lacking. Here it is established that chromosomal aberrations detected in bone marrow cells by chromosome painting are induced in all mice at an approximately 2-fold greater frequency in chr 2 by comparison with chrs 1 and 3 at 24 and 48 h following in vivo whole-body X-irradiation. Long-term follow up studies (to 15 months post-irradiation) indicated that chromosomal hypersensitivity is accounted for largely by the existence of hot-spots for aberration formation on sensitive chromosomes. Analysis of clonal developments suggested that chr 2 aberrant clones are selected for entry into the proliferating bone marrow cell compartment in preference to cells with other aberrations and that these clones in general have a higher proliferative potential. However, neither the induction of chr 2 aberrations nor the presence of a chr 2 aberrant clone specifically predict the development of AML in an individual irradiated mouse. Nonetheless these events or sub-groups of these events are necessary for AML development.

The Gorlin syndrome, or naevoid basal-cell carcinoma syndrome (NBCS) is an autosomal dominant disease. It has been suspected for long that this cancer prone disease (multiple basal-cell carcinomas; other malignant or benign proliferations) is a chromosome instability syndrome. We previously reported a lengthening in the cell cycle of lymphocytes from two patients with NBCS. With a larger sample (n = 7), we confirm this disease to be a chromosome instability syndrome, although clearly, expression of this characteristic can vary between patients: (1) spontaneous chromatid breaks occurred more often in a subset of the patients; (2) spontaneous micronuclei were found more frequently in NBCS than in the controls; (3) we confirm the cell cycle to be affected in this disease. As these results were obtained on lymphocytes--a cell lineage not affected in NBCS manifestations--the chromosome instability we found would appear to be part of the general condition of this syndrome.

SUMMARYPrevious studies have shown that the mouseXchromosomal locus,Xce, which causes non-randomXchromosome inactivation, is closely linked to the Is(X; 7)CtX-autosome translocation. This has placed it either nearTaon one side of the breakpoint or nearjpon the other. Linkage tests withMovbrandTanow demonstrate that the locus in fact lies close toTa. The data also provide genetic evidence which establish that the C3H/HeHXchromosome carries theXceaallele of this gene and the JU/FaCt and C57BL/GoHXchromosomes carry theXceballele, and further suggest that theX-linked modification of the heterozygous phenotypes ofX-linked genes observed by various other investigators are all attributable to differences at theXcelocus. Evidence of a maternal influence uponMovbrphenotypes is also presented. This appears to operate independently of theX-inactivation process, probably through an effect of differing levels of copper in the milk in early life upon the mutant coat colour in the young.

Two new genetic methods for detecting autosomal non-disjunction and chromosome loss in mice are described. Both methods involve the use of marker genes and Robertsonian translocations, the latter present only in tester parents, to detect events in chromosomally normal mice. With the Rb method, the tester parent carries one or more Robertsonian translocations heterozygously; with the MBH method the tester parent carries two Robertsonian translocations showing monobrachial homology. The high rates of meiotic non-disjunction in the tester mice provide gametes with specific extra or missing chromosomes which, at fertilization, can allow the survival of a proportion of the zygotes lacking or carrying an extra specific chromosome from tested chromosomally normal parents. The Rb method has been assessed for X-ray-induced chromosome 1 loss and non-disjunction in mature oocytes and also for such chromosome 1 loss from the maternal pronuclei of 1-cell zygotes. The MBH method has been assesses for X-ray-induced chromosome 1 loss in male postmeiotic cells ad for non-disjunction in spermatocytes. Both methods proved effective in detecting chromosome 1 loss. A single case of the much rarer non-disjunctional event was also found. As applied, both methods compared favourably with the numerical sex chromosome anomaly (NSA) method and have considerable potential for further development.

We tested whether modulation of the CNS-tumor microenvironment by delivery of IFN-α-transduced dendritic cells (DCs: DC-IFN-α) would enhance the therapeutic efficacy of peripheral vaccinations with cytokine-gene transduced tumor cells. Mice bearing intracranial GL261 glioma or MCA205 sarcoma received peripheral immunizations with corresponding irradiated tumor cells engineered to express IL-4 or GM-CSFs, respectively, as well as intratumoral delivery of DC-IFN-α. This regimen prolonged survival of the animals and induced tumor-specific CTLs that expressed TRAIL, which in concert with perforin and Fas ligand (FasL) was involved in the tumor-specific CTL activity of these cells. The in vivo antitumor activity associated with this approach was abrogated by administration of neutralizing mAbs against TRAIL or FasL and was not observed in perforin−/−, IFN-γ−/−, or FasL−/− mice. Transduction of the tumor cells with antiapoptotic protein cellular FLIP rendered the gene-modified cells resistant to TRAIL- or FasL-mediated apoptosis and to CTL killing activity in vitro. Furthermore, the combination therapeutic regimen was ineffective in an intracranial cellular FLIP-transduced MCA205 brain tumor model. These results suggest that the combination of intratumoral delivery of DC-IFN-α and peripheral immunization with cytokine-gene transduced tumor cells may be an effective therapy for brain tumors that are sensitive to apoptotic signaling pathways.