Development of an intrabiliary MR imaging-monitored local agent delivery technique: a feasibility study in pigs.

PURPOSE: To investigate the feasibility of using magnetic resonance (MR) imaging to monitor intrabiliary delivery of motexafin gadolinium (MGd) into pig common bile duct (CBD) walls. MATERIALS AND METHODS: Animal studies were approved by the Institutional Animal Care and Use Committee. Initially, human cholangiocarcinoma cells were treated with various concentrations of MGd, a compound serving as a T1-weighted MR imaging contrast agent, chemotherapy drug, and cell marker. These cells were then examined by means of confocal microscopy to confirm the intracellular uptake of MGd. In addition, an MGd/trypan blue mixture was locally infused into CBD walls of six cadaveric pigs using a microporous balloon catheter. CBDs of six pigs were infused with saline to serve as controls. Ex vivo T1-weighted MR imaging of these CBDs was performed. For in vivo technical validation, the microporous balloon catheter was placed in the CBD by means of a transcholecytic access to deliver MGd/trypan blue into CBD walls of six living pigs. T1-weighted images were obtained with both a surface coil and an intrabiliary MR imaging guidewire, and contrast-to-noise ratios of CBD walls before and after MGd/trypan blue infusions were compared in the two groups by means of paired t test, with subsequent histologic analysis to confirm the penetration and distribution of the MGd/trypan blue agent into CBD walls. RESULTS: In vitro experiments confirmed uptake of MGd by human cholangiocarcinoma cells. The ex vivo experiments demonstrated the penetration of MGd/trypan blue into the CBD walls. The in vivo experiment confirmed the uptake of MGd/trypan blue, showing an increased contrast-to-noise ratio for the CBD after administration of the mixture, compared with images obtained prior to MGd/trypan blue administration (11.6 ± 4.2 [standard deviation] vs 5.7 ± 2.8; P = .04). Histologic results depicted the blue dye stains and red fluorescence of MGd in CBD walls, confirming the imaging findings. CONCLUSION: It is feasible to use MR imaging to monitor the penetration of locally delivered MGd into pig CBD walls.

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume.

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof-of-concept, we characterized high-speed PAFC capability to monitor the clearance of three dyes (Indocyanine Green [ICG], Methylene Blue [MB], and Trypan Blue [TB]) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including Crystal Violet and Brilliant Green were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, and transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is safe for human, and its applications for studying the liver function are further highlighted.

Pluronic enhances the robustness and reduces the cell attachment of mammalian cells.

The addition of the non-ionic surfactant, Pluronic F-68, to serum-free CHO cultures causes multi-functional effects that enhance cell yield in agitated cultures and reduce cell adhesion in stationary cultures. Three independent CHO cell lines were subjected to high liquid shear in assay systems that either included or excluded a liquid-gas interface. In the absence of Pluronic, there was a loss in cell viability in either assay system, although there was an intrinsic variability in sensitivity of the cell lines to shear damage. Supplementation with Pluronic prevented loss of cell viability, indicating protection in either a gas sparged or bubble-free environment. However, we found no evidence of long-term protection of cells once Pluronic was removed. Pluronic was capable of repairing trypsin-damaged cells as evidenced by enhanced growth, reduced membrane porosity, and improved robustness under liquid shear. The proportion of adherent cells was reduced to a minimal level by the presence of Pluronic although its effect was rapidly reversible with a high proportion (70%) of adherent cells observed within a few culture passages of its removal. The observed effects of Pluronic on these cultures are compatible with a mechanism in which the polymer forms a protective layer on the cell membrane, which has a significantly lower hydrophobicity.

A Novel Approach for Therapeutic Delivery to the Rodent Pancreas Via Its Arterial Blood Supply.

OBJECTIVES: Endovascular techniques can now access the arterial blood supply of the pancreas in humans to enable therapeutics to reach the gland in high concentrations while concurrently avoiding issues related to non-targeted delivery. However, there is no way to replicate this in small animals. In a rat model, we therefore developed a novel non-terminal technique to deliver therapeutics to different regions of the pancreas, via its arterial blood supply. METHODS: In female Wistar rats, selective branches of the celiac artery were temporarily ligated, depending on the region of the pancreas being targeted. Trypan blue dye was then administered as a surrogate marker for a therapeutic agent, via the celiac artery, and its staining/distribution throughout the pancreas determined. Postoperatively, animals were monitored daily, and serum was evaluated for markers of pancreatitis, liver, and metabolic function. RESULTS: Using this technique, we could selectively target the head, body/tail, or entire gland of the pancreas, via its arterial blood supply, with minimal nontarget staining. Following the procedure, all animals recovered with no evidence of pancreatitis or liver/metabolic dysfunction. CONCLUSIONS: Our study demonstrates a novel technique that can be used to selectively deliver therapeutics directly to the rat pancreas in a safe manner with full recovery of the animal.

Assembled microneedle arrays enhance the transport of compounds varying over a large range of molecular weight across human dermatomed skin.

In this study, we demonstrate the feasibility to use microneedle arrays manufactured from commercially available 30G hypodermal needles to enhance the transport of compounds up to a molecular weight of 72 kDa. Piercing of human dermatomed skin with microneedle arrays was studied by Trypan Blue staining on the SC side of the skin and transepidermal water loss measurements (TEWL). Passive transport studies were conducted with Cascade Blue (CB, Mw 538), Dextran-Cascade Blue (DCB, Mw 10 kDa), and FITC coupled Dextran (FITC-Dex, Mw 72 kDa). Microneedle arrays with needle lengths of 900, 700 and 550 micro m are able to pierce dermatomed human skin as evident from (a) the appearance of blue spots on the dermal side of the skin after Trypan Blue treatment and (b) elevated TEWL levels after piercing compared to non-treated human dermatomed skin. Microneedles with a length of 300 micro m did not pierce human skin in vitro. Transport studies performed with model compounds ranging from 538 Da to 72 kDa revealed that pretreatment with microneedle arrays enhanced the transport across dermatomed human skin. However, some degradation was also observed for FITC-Dex and DCB. We conclude that assembled microneedle arrays can be used to deliver compounds through the skin up to a molecular weight of at least 72 kDa.

Etoposide-induced cytotoxicity in two human T-cell leukemic lines: delayed loss of membrane permeability rather than DNA fragmentation as an indicator of programmed cell death.

Features of the apoptotic response evident in glucocorticoid-treated thymocytes are not uniformly observed in cell lines exposed to anticancer drugs. The significance of such variation has been assessed by monitoring molecular and cellular processes induced by etoposide (VP-16) in the human lymphoblastoid T-cell lines CCRF-CEM (CEM) and MOLT-4 contrasted, where appropriate, with those induced by necrotizing injury. Cytotoxic concentrations of the drug were determined to be 5-100 microM on the basis of tetrazolium reduction assay. The two lines were equally sensitive to VP-16; no difference in concentration of drug which inhibited cell growth by 50% with respect to control (i.e., drug free) cultures was apparent irrespective of exposure times from 3-72 h. DNA strand breaks were evident in both populations within 3 h of exposure to VP-16. Morphological change, assessed microscopically, involving nuclear condensation and cell shrinkage was qualitatively and quantitatively similar in VP-16-treated CEM and MOLT-4 cells. Flow cytometric analysis indicated that the G2/M fraction of the randomly dividing MOLT-4 population was approximately one-third that of CEM cells, but each line exhibited a decrease in this fraction 3-6 h after treatment. Despite these similarities, marked differences in the response to VP-16 were evident in the two populations. Internucleosomal fragmentation, detected electrophoretically 3 h after treatment in DNA isolated from CEM cells, was not detected under any condition in MOLT-4 DNA. Apoptotic bodies, also evident within 3 h of VP-16 treatment of CEM cells, were not readily apparent in MOLT-4 cells under the same conditions. Treatment causing necrosis resulted in trypan blue uptake within 1 h in a similar high proportion of cells from both lines. Exposure to VP-16 resulted in such a loss of membrane integrity by 6 h in CEM cells, while change in this parameter occurred only after 24 h in the case of MOLT-4 cells. The findings indicate a wide scope of difference in apoptotic response and suggest delayed loss of membrane permeability, rather than DNA fragmentation, as the clearest indicator of programmed cell death in these populations.

Grooved and nongrooved clear corneal incisions in phacoemulsification: permeability study.

PURPOSE: To detect the inflow of trypan blue through grooved and nongrooved sutureless self-sealing clear corneal incisions at the end of phacoemulsification as compared to a control group. METHODS: A prospective randomized masked trial considered 52 eyes randomized into 3 groups in which phacoemulsification was performed: group A, nongrooved incisions; group B, grooved incisions; and group C, controls. By the end of each surgery, trypan blue was instilled on the ocular surface in groups A and B and rinsed out after 2 minutes. A sample of the anterior chamber content was collected and analyzed by high-performance liquid chromatography to identify and quantify the trypan blue concentration. The presence of trypan blue was expressed as a specific single peak graphic image. The mean areas of these peaks were used to assess the groups using a nonparametric Mann-Whitney test. RESULTS: There was a statistically significant difference between the nongrooved incisions group and the control group (p = 0.0448). No significant difference was observed between group B (grooved incision) and controls (p = 0.1800). CONCLUSIONS: Trypan blue was detected in the anterior chamber when nongrooved clear corneal incision was used. There was no trypan blue detection in the group with grooved clear corneal main incisions.

Superiority of the two-layer method before islet isolation confirmed by in vivo viability assessment.

BACKGROUND: Although the outcome of islet transplantation has improved, there remains a major obstacle in isolating viable islets from prolonged preserved pancreas. We previously reported that the two-layer cold storage method (TLM) improved the yield and in vitro function. In this study, we performed in vivo accurate functional analyses of islets from TLM-preserved pancreas and investigated pancreatic duct cell viability, which may critically affect islet isolation. METHODS: Rat islets isolated from fresh pancreas (group 1), after preservation in the University of Wisconsin (UW) solution (group 2) or by the TLM (group 3), were examined by assessing islet yields, stimulation indices, cure rates after transplantation to diabetic nude mice, and trypan blue uptake of pancreatic duct cells. RESULTS: TLM significantly improved the islet yield compared with UW cold storage. The cure rates after transplantation were 100%, 0%, and 80% for groups 1, 2, and 3, respectively. This indicates that islet viability was well maintained even after 24 hr of TLM preservation. The percentages of nonviable duct cells were 4.1%+/-1.9%, 48.3%+/-8.0%, and 26.1%+/-21.4% in groups 1, 2, and 3, respectively, showing that the TLM was superior to UW as seen by this duct cell viability assessment. CONCLUSIONS: The TLM used for pancreas preservation before islet isolation results in excellent islet function in addition to improved islet yield comparable to freshly isolated islets. The underlying mechanism may be duct cell viability maintained during TLM preservation. Therefore the TLM is an excellent preservation technique for isolating sufficient numbers of highly viable islets.

Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents.

OBJECT: Clinical application of the convection-enhanced delivery (CED) technique is currently limited by low infusion speed and reflux of the delivered agent. The authors developed and evaluated a new step-design cannula to overcome present limitations and to introduce a rapid, reflux-free CED method for future clinical trials. METHODS: The CED of 0.4% trypan blue dye was performed in agarose gel to test cannula needles for distribution and reflux. Infusion rates ranging from 0.5 to 50 microl/minute were used. Agarose gel findings were translated into a study in rats and then in cynomolgus monkeys (Macacafascicularis) by using trypan blue and liposomes to confirm the efficacy of the reflux-free step-design cannula in vivo. Results of agarose gel studies showed reflux-free infusion with high flow rates using the step-design cannula. Data from the study in rats confirmed the agarose gel findings and also revealed increasing tissue damage at a flow rate above 5-microl/minute. Robust reflux-free delivery and distribution of liposomes was achieved using the step-design cannula in brains in both rats and nonhuman primates. CONCLUSIONS: The authors developed a new step-design cannula for CED that effectively prevents reflux in vivo and maximizes the distribution of agents delivered in the brain. Data in the present study show reflux-free infusion with a constant volume of distribution in the rat brain over a broad range of flow rates. Reflux-free delivery of liposomes into nonhuman primate brain was also established using the cannula. This step-design cannula may allow reflux-free distribution and shorten the duration of infusion in future clinical applications of CED in humans.

Kynurenine pathway inhibition reduces neurotoxicity of HIV-1-infected macrophages.

The AIDS dementia complex (ADC) is a consequence of excessive immune activation driven at least in part by systemic HIV infection and probably brain infection. Quinolinic acid (QUIN) is a neurotoxic tryptophan metabolite produced by macrophages in response to stimulation with cytokines or infection with HIV-1. Consequently it has been implicated in ADC pathogenesis. However, macrophages infected with HIV-1 synthesize numerous neurotoxic substances. Therefore we conducted experiments using human fetal brain tissue to determine the relative importance of QUIN as a neurotoxin in ADC. Human macrophages were infected with HIV-1 in vitro using a viral isolate from a demented patient. 6-Chloro-D-tryptophan, an inhibitor of QUIN biosynthesis, was added to half the macrophage cultures to block formation of QUIN. Supernatants containing QUIN (SQpos) or in which QUIN biosynthesis had been inhibited (SQneg) were then added to human fetal brain aggregate cultures. Toxicity was evaluated using lactate dehydrogenase efflux, trypan blue exclusion, immunohistochemistry, image analysis, and electron microscopy. Each technique showed a reduction of toxicity in SQneg-treated cultures. These studies confirm the significance of QUIN as a neurotoxin in ADC and suggest that neuroprotective strategies may have a place in the treatment of this disease.

The effect of ouabain (inhibitor of an ATP-dependent Na+/K+ pump) on the pancreas graft during preservation by the two-layer method.

To clarify how synthesized ATP is utilized to maintain cellular integrity during preservation by the two-layer method, we examined the effect of ouabain, inhibitor of ATP dependent Na+/K+ pump, on ATP tissue levels, graft weight, and vascular endothelial cells during 48 hr preservation by the two-layer method and pancreatic tissue perfusion and graft survival after transplantation in a canine model. Ouabain treatment did not affect ATP production but prevented its utilization by the sodium pump, and actually significantly increased ATP levels and decreased the weight loss of the graft. In addition, ouabain caused a significant increase of nuclear trypan-blue staining in vascular endothelial cells and a significant decrease of pancreatic tissue perfusion at reperfusion. Consequently, the grafts did not survive. We conclude that the two-layer method allows sufficient synthesis of ATP to drive the sodium pump and maintains membrane integrity of parenchymal cells and vascular endothelial cells, thus extending the period of preserved pancreatic viability.

Extending the margin of safety of preservation period for resuscitation of ischemically damaged pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C with thromboxane A2 synthesis inhibitor OKY046.

We have shown that 5-hr preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C allows ATP synthesis and makes it possible to resuscitate a canine pancreas subjected to 90 min of warm ischemia. However, 8 hr of preservation using this method caused a disturbance of vascular microcirculation and did not resuscitate the grafts. The aim of this study was to examine the effect of thromboxane A2 synthesis inhibitor OKY046 on vascular endothelial cells and ATP tissue levels of canine pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C, and vascular microcirculation and pancreas viability after transplantation. Graft viability was judged by graft survival following autotransplantation. ATP tissue levels were measured by high-performance liquid chromatography at the end of preservation. Viability of the vascular endothelial cells was judged using nuclear trypan blue uptake of the graft after preservation. Pancreatic tissue perfusion was measured using an H2 clearance technique after reperfusion. Pancreas grafts subjected to 90 min of warm ischemia were not viable (0/5). However, 5-hr preservation made it possible to recover the pancreas (5/5); 8-hr preservation was not successful (0/3). ATP tissue levels after 5-hr and 8-hr preservation were 9.40+/-2.09 and 7.37+/-1.06 micromol/g dry weight, respectively, and OKY046 did not affect ATP synthesis during 8-hr preservation (8.44+/-0.92 micromol/g dry weight). The percentage of nuclear trypan blue uptake of endothelial cells in 8-hr-preserved grafts was 37.6+/-11.6% and was significantly higher than the value in 5-hr-preserved grafts (5.0+/-3.0%; P<0.01). However, OKY046 significantly reduced trypan blue uptake in 8-hr-preserved grafts (8.2+/-3.6%; P<0.01). Pancreatic tissue perfusion in 8-hr-preserved grafts after 2 hr of reperfusion was 28.5+/-7.5 ml/min/100 g, and was significantly lower than the value in 5-hr-preserved grafts (57.1+/-4.4 ml/ min/100 g; P<0.01), but OKY046 dramatically improved pancreatic tissue perfusion (97.1+/-14.6 ml/min/100 g; P<0.01). As a consequence, 8-hr-preserved grafts were resuscitated (4/5). We conclude that OKY046 protects the vascular endothelium during preservation by the two-layer method at 20 degrees C and consequently improves vascular microcirculation on reperfusion. Together with ATP synthesis, which is essential for repairing damaged cells, the canine pancreas graft subjected to 90 min of warm ischemia is resuscitated during 8-hr preservation by the two-layer method at 20 degrees C. This method holds promise for pancreas-kidney transplantation from cardiac arrest donors.

Some histological and metabolic properties of an isolated perfused rat brain preparation with special reference to monoamine metabolism.

A method is described for perfusing the rat head which results in perfusion of the brain, its covering and the bony skull only. Dye and latex injection studies showed that there was no perfusion of extracranial tissue and thus it was not necessary to remove the lower jaw and facial tissues as described by previous authors. Perfusion with fluorescein demonstrated that the whole brain was perfused. Light microscopy after two hours of perfusion revealed no deterioration in brain-structure. Electron microscopy showed a small increase in the extracellular space and the perivascular space with good preservation of subcellular organelles. Glucose and acetoacetate removal were very similar to those reported for the adult rat in vivo as measured by arterio-venous differences. The concentrations of 5-hydroxytryptamine, dopamine and noradrenaline are maintained at values close to those in vivo during a 2-hr perfusion with a basal medium and the preparation will maintain linear rates of 5-hydroxytryptamine synthesis for 2 h when the medium contains L-tryptophan and tranylcypromine, at rates similar to those measured in vivo. The rate of uptake of L-tryptophan into the brain is similar to that reported after L-tryptophan loading in vivo. The histological and metabolic properties of the preparation are close to those observed in vivo. The method could provide a way in which monoamine metabolism in particular could be studied using an experimental model closer in its properties to the in vivo situation than tissue preparations such as slices or homogenates.

The effects of the fluorinated pyrimidines FUR, FUdR, FUMP, and FdUMP on human Tenon's fibroblasts.

5-Fluorouracil (5-FU) has effectively inhibited fibroblast proliferation to prevent scar formation and bleb failure after glaucoma filtering surgery. To identify more potent but less toxic antiproliferative drugs, the authors studied cell attachment and proliferation of 5-FU metabolites: 5-fluorouridine (FUR), 5-fluorodeoxyuridine (FUdR), 5-fluorouridine-5'-monophosphate (FUMP), and 5-fluorodeoxyuridine-5'-monophosphate (FdUMP) on human Tenon's fibroblasts in vitro.

Protective effects of the calcium antagonists diltiazem and TA3090 against hepatic injury due to hypoxia.

Recent work has shown that dihydropyridine-type calcium channel blockers such as nitrendipine protect against ischemic liver damage in the rat in vivo (Thurman RG, Apel E and Lemasters JJ, J Cardiovasc Pharmacol 12: S113-S116, 1988), suggesting that calcium antagonists may have clinical value in preventing ischemic and hypoxic hepatic injury. This study was designed to examine the effects of two benzothiazepine-type calcium channel blockers, diltiazem and TA3090, in the hypoxic perfused rat liver. Livers were isolated and perfused briefly with oxygen-saturated buffer, followed by perfusion for 80 min with nitrogen-saturated buffer with diltiazem or TA3090 (20-200 microM), and concluding with 20 min of perfusion with oxygen-saturated buffer. In control preparations, maximal lactate dehydrogenase (LDH) release into effluent perfusate following hypoxia averaged about 1100 U/L. Diltiazem and TA3090 decreased LDH release at all concentrations studied; both drugs were most effective at the 100 microM concentration (71 and 73% inhibition, respectively). Oxygen uptake by control livers decreased 78% following hypoxia; diltiazem and TA3090 reduced this effect markedly, with maximal effectiveness again observed with 100 microM (O2 uptake was decreased by 22% with 100 microM diltiazem and by only 9% with 100 microM TA3090). Histological examination for nuclear uptake of the vital dye trypan blue revealed necrosis of parenchymal cells along with cell shrinking and consequent expansion of the sinusoids in control livers. Perfusion with diltiazem markedly reduced parenchymal cell death but did not alter the pattern of cell damage observed. In contrast, livers perfused with TA3090 during hypoxia had virtually no parenchymal cell damage, although moderate damage to nonparenchymal cells in the sinusoids occurred. The difference in mechanisms responsible for the phenomena which occur with diltiazem and TA3090 is not completely understood; however, these and other calcium antagonists clearly have powerful hepatoprotective effects against ischemia and hypoxia.

Antioxidant enzymes are elevated in dimethylbenz[a]anthracene-induced neoplastic murine keratinocytes containing an active rasHa oncogene.

Antioxidant enzyme activities and peroxidation potential were measured in primary mouse keratinocytes and neoplastic keratinocytes containing an active rasHa oncogene. In neoplastic cell lines, SP-1 and 308, the activities of Cu, Zn-superoxide dismutase, catalase, and glutathione transferase were significantly elevated. The peroxidation potential was lower in cell homogenates prepared from neoplastic keratinocytes than in those prepared from normal keratinocytes. Consistently, the neoplastic 308 cell line was found to be more resistant than the normal keratinocytes to cytotoxicity induced by UV-B irradiation. The present study suggests that the enhanced antioxidant defense system protects the initiated cells from UV-B-induced oxidative stress, and that the enhanced enzymic antioxidant defense system is potentially a mechanism favoring the selective growth of neoplastic keratinocytes.

Development of an intact blood-brain barrier in brain tissue transplants is dependent on the site of transplantation.

Transplantation of fetal septal forebrain tissue was performed to the anterior chamber of the eye, or intracranially to the rostral hippocampal formation in rats, to evaluate the impact of transplantation site on the development of an intact blood-brain barrier (BBB). The tissue was studied at 1,2,3, and 4 wk following transplantation by means of intravenous injection of Trypan blue, which is a vital stain not normally penetrating the BBB, as well as with an antibody specifically directed against the rat BBB, SM171. In the intraocular septal transplants, there was a significant leakage of Trypan blue 1 wk postgrafting, associated with a few laminin-immunoreactive blood vessels that did not contain any SM17I-immunoreactivity. However, at 2 wk postgrafting, the intraocular grafts exhibited an extensive plexus of thin-walled blood vessels expressing SMI71 immunoreactivity and no Trypan blue leakage. Thus, it appeared that a BBB had developed to some degree by 2 wk postgrafting in oculo. In the intracranial grafts, on the other hand, Trypan blue leakage could be seen as long as 3 wk postgrafting, and a dense plexus of blood vessels with SMI71 immunoreactivity was first seen at 4 wk postgrafting. Thus, the development of Trypan blue impermeability was delayed with 1 to 2 wk in the intracranial versus the intraocular grafts. Control experiments using psychological stress in adult rats as a means to transiently disrupt the BBB revealed that an increase in Trypan blue leakage correlated well with the disappearance of SMI71 immunoreactivity. Taken together, these studies demonstrate that the site of transplantation can influence the development of an intact BBB in neural tissue grafts.

Lipopolysaccharide induces Ca(2+)-independent nitric oxide synthase activity in rat gastric mucosal cells.

Ca(2+)-independent nitric oxide synthase was detected in gastric mucosal cells isolated from rats injected 4 h previously with Escherichia coli lipopolysaccharide (3 mg/kg i.v.). Induced nitric oxide synthase was located in an elutriated cell fraction of intermediate size which contained epithelial cells, but was absent from the parietal cell fraction. Administration of dexamethasone (2 mg/kg i.p.) 1 h before lipopolysaccharide inhibited the appearance of Ca(2+)-independent nitric oxide synthase, and prevented the observed reduction in cell viability (trypan blue exclusion). Ca(2+)-independent nitric oxide synthase activity can thus be induced in certain cells of the gastric mucosa, and may contribute to gastric pathologies where there is activation of the immune system.

Triphenyltin acetate-induced cytotoxicity and CD4(+) and CD8(+) depletion in mouse thymocyte primary cultures.

Organotin compounds (OTs) find application worldwide as catalysts, stabilizers and biocides. Triphenyltin derivatives (TPs), including the fungicide triphenyltin acetate (TPTA), are OTs mostly used in our country. Some OTs were proved to be immunotoxic and in this paper the cytotoxicity, the possible selective activity upon definite lymphocyte subsets as well as the antiproliferative effect of TPTA was investigated in vitro by using primary cultures of mouse thymocytes. TPTA (5, 10 and 25 microM) was cytotoxic to these cells, as demonstrated by the significant (P<0.05) reduction of the cell viability percentage (trypan blue dye exclusion test), the neutral red uptake and the reduction of tetrazolium salts to formazan products (MTT assay). These overt effects were already noticed after 4 h of exposure to TPTA. The fungicide otherwise significantly reduced, after 24 h of incubation, the percentage of mature single positive thymocytes, particularly the CD4(+)/CD8(-) one. Finally, a significative dose-dependent inhibition of the T-cell mitogen-induced cell proliferation was observed in thymocytes exposed to 1 and 8 microM TPTA. These results are indicative of the TPTA immunotoxic properties, according to previous published reports concerning the in vitro and in vivo toxicity of some di- and triorganotin compounds.