Live-cell imaging reveals the relative contributions of antigen-presenting cell subsets to thymic central tolerance.

Both medullary thymic epithelial cells (mTEC) and dendritic cells (DC) present tissue-restricted antigens (TRA) to thymocytes to induce central tolerance, but the relative contributions of these antigen-presenting cell (APC) subsets remain unresolved. Here we developed a two-photon microscopy approach to observe thymocytes interacting with intact APCs presenting TRAs. We find that mTECs and DCs cooperate extensively to induce tolerance, with their relative contributions regulated by the cellular form of the TRA and the class of major histocompatibility complex (MHC) on which antigen is presented. Even when TRA expression is restricted to mTECs, DCs still present self-antigens at least as frequently as mTECs. Notably, the DC subset cDC2 efficiently acquires secreted mTEC-derived TRAs for cross-presentation on MHC-I. By directly imaging interactions between thymocytes and APCs, while monitoring intracellular signaling, this study reveals that distinct DC subsets and AIRE+ mTECs contribute substantially to presentation of diverse self-antigens for establishing central tolerance.

Sex-specific and lasting effects of a single course of antenatal betamethasone treatment on human placental 11ß-HSD2.

INTRODUCTION: We have previously shown that even a single course of antenatal betamethasone (BET) as an inductor for lung maturity reduces birth weight and head circumference. Moreover, animal studies link BET administration to alterations of the hypothalamic-pituitary-adrenal-gland-axis (HPA). The unhindered development of the fetal HPA axis is dependent on the function and activity of 11ß-hydroxysteroiddehydrogenase type 2 (11ß-HSD2), a transplacental cortisol barrier. Therefore, we investigated the effects of BET on this transplacental barrier and fetal growth. METHODS: Pregnant women treated with a single course of BET between 23 + 5 to 34 + 0 weeks of gestation were compared to gestational-age-matched controls. Placental size and neonatal anthropometrics were taken. Cortisol and ACTH levels were measured in maternal and umbilical cord blood samples. Placental 11ß-hydroxysteroiddehydrogenase type 1 (11ß-HSD1) protein levels and 11ß-HSD2 protein and activity levels were determined. Parameters were analyzed independent of sex, and in subgroups divided by gender and gestational age. RESULTS: In term born females, BET administration was associated with reduced head circumference and decreased 11ß-HSD2 protein levels and enzyme activity. Males treated with BET, especially those born prematurely, showed increased 11ß-HSD2 protein levels. CONCLUSION: A single course of BET alters placental glucocorticoid metabolism in a sex-specific manner. Decreased 11ß-HSD2 levels in term born females may lead to an increased placental transfer of maternal cortisol and therefore result in a reduced head circumference and a higher risk for altered stress response in adulthood. Further research is needed to conclude the significance of increased 11ß-HSD2 levels in males.

The importance of selecting the right internal control gene to study the effects of antenatal glucocorticoid administration in human placenta.

RT-qPCR requires a suitable set of internal control genes (ICGs) for an accurate normalization. The usefulness of 7 previously published ICGs in the human placenta was analyzed according to the effects of betamethasone treatment, sex and fetal age. Raw RT-qPCR data of the ICGs were evaluated using published algorithms. The algorithms revealed that a reliable normalization was achieved using the geometrical mean of PPIA, RPL19, HMBS and SDHA. The use of a different subset ICGs out of the 7 investigated, although not statistically affected by the conditions, biased the results, as demonstrated through changes in expression of glucocorticoid receptor (NR3C1) mRNA as a target gene.

Effects of maternal dexamethasone treatment early in pregnancy on glucocorticoid receptors in the ovine placenta.

The effects of endogenous cortisol on binucleate cells (BNCs), which promote fetal growth, may be mediated by glucocorticoid receptors (GRs), and exposure to dexamethasone (DEX) in early pregnancy stages of placental development might modify this response. In this article, we have investigated the expression of GR as a determinant of these responses. Pregnant ewes carrying singleton fetuses (n = 119) were randomized to control (2 mL saline/ewe) or DEX-treated groups (intramuscular injections of 0.14 mg/kg ewe weight per 12 hours) at 40 to 41 days of gestation (dG). Placental tissue was collected at 50, 100, 125, and 140 dG. Total glucocorticoid receptor protein (GRt) was increased significantly by DEX at 50 and 125 dG in females only, but decreased in males at 125 dG as compared to controls. Glucocorticoid receptor α (GRα) protein was not changed after DEX treatment. Three BNC phenotypes were detected regarding GRα expression (++, +-, --), DEX increased the proportion of (++) and decreased (--) BNC at 140 dG. Effects were sex- and cell type dependent, modifying the responsiveness of the placenta to endogenous cortisol. We speculate that 3 maturational stages of BNCs exist and that the overall activity of BNCs is determined by the distribution of these 3 cell types, which may become altered through early pregnancy exposure to elevated glucocorticoids.

When the tumour is not the culprit: avascular necrosis of the hip in a patient with castration-resistant prostate cancer.

Avascular necrosis (avn) of the hip is a well-documented side effect of corticosteroid therapy, but it has also been described as a complication of radiation and chemotherapy. Many prostate cancer patients undergo treatment with all three of those therapeutic modalities, and yet reported cases of avn of the hip in prostate cancer patients are rare. Symptoms that might potentially alert physicians to this complication are nonspecific and may be attributed to cancer progression, in particular to progressive bone metastasis.Here, we report on a 79-year-old man diagnosed with castration-resistant prostate cancer whose diagnosis of avn of the hip was confounded by his underlying malignancy. We discuss risk factors and diagnostic clues in this differential diagnosis of acute hip pain in patients with castration-resistant prostate cancer. Physicians might maintain a high index of suspicion for avn of the hip in prostate cancer patients presenting with new-onset hip pain. Surgical intervention may help to prevent the appearance of avn-associated pain and the negative impact of advanced avn on overall quality of life.

Sprouty 2 binds ESCRT-II factor Eap20 and facilitates HIV-1 gag release.

The four ESCRT (endocytic sorting complexes required for transport) complexes (ESCRT-0, -I, -II, and -III) normally operate sequentially in the trafficking of cellular cargo. HIV-1 Gag trafficking and release as virus-like particles (VLPs) require the participation of ESCRTs; however, its use of ESCRTs is selective and nonsequential. Specifically, Gag trafficking to release sites on the plasma membrane does not require ESCRT-0 or -II. It is known that a bypass of ESCRT-0 is achieved by the direct linkage of the ESCRT-I component, Tsg101, to the primary L domain motif (PTAP) in Gag and that bypass of ESCRT-II is achieved by the linkage of Gag to ESCRT-III through the adaptor protein Alix. However, the mechanism by which Gag suppresses the interaction of bound ESCRT-I with ESCRT-II is unknown. Here we show (i) that VLP release requires the steady-state level of Sprouty 2 (Spry2) in COS-1 cells, (ii) that Spry2 binds the ESCRT-II component Eap20, (iii) that binding Eap20 permits Spry2 to disrupt ESCRT-I interaction with ESCRT-II, and (iv) that coexpression of Gag with a Spry2 fragment that binds Eap20 increases VLP release. Spry2 also facilitated release of P7L-Gag (i.e., release in the absence of Tsg101 binding). In this case, rescue required the secondary L domain (YPX(n)L) in HIV-1 Gag that binds Alix and the region in Spry2 that binds Eap20. The results identify Spry2 as a novel cellular factor that facilitates release driven by the primary and secondary HIV-1 Gag L domains.

Epigenetic memory in induced pluripotent stem cells.

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

Reductive isolation from bone marrow and blood implicates common lymphoid progenitors as the major source of thymopoiesis.

Ongoing thymopoiesis requires continual seeding from progenitors that reside within the bone marrow (BM), but the identity of the most proximate prethymocytes has remained controversial. Here we take a comprehensive approach to prospectively identify the major source of thymocyte progenitors that reside within the BM and blood, and find that all thymocyte progenitor activity resides within a rare Flk2(+)CD27(+) population. The BM Flk2(+)CD27(+) subset is predominantly composed of common lymphoid progenitors (CLPs) and multipotent progenitors. Of these 2 populations, only CLPs reconstitute thymopoiesis rapidly after intravenous injection. In contrast, multipotent progenitor-derived cells reconstitute the thymus with delayed kinetics only after they have reseeded the BM, self-renewed, and generated CLPs. These results identify CLPs as the major source of thymocyte progenitors within the BM.

Developing a methodology for three-dimensional correlation of PET-CT images and whole-mount histopathology in non-small-cell lung cancer.

BACKGROUND: Understanding the three-dimensional (3D) volumetric relationship between imaging and functional or histopathologic heterogeneity of tumours is a key concept in the development of image-guided radiotherapy. Our aim was to develop a methodologic framework to enable the reconstruction of resected lung specimens containing non-small-cell lung cancer (NSCLC), to register the result in 3D with diagnostic imaging, and to import the reconstruction into a radiation treatment planning system. METHODS AND RESULTS: We recruited 12 patients for an investigation of radiology-pathology correlation (RPC) in nsclc. Before resection, imaging by positron emission tomography (PET) or computed tomography (CT) was obtained. Resected specimens were formalin-fixed for 1-24 hours before sectioning at 3-mm to 10-mm intervals. To try to retain the original shape, we embedded the specimens in agar before sectioning. Consecutive sections were laid out for photography and manually adjusted to maintain shape. Following embedding, the tissue blocks underwent whole-mount sectioning (4-mum sections) and staining with hematoxylin and eosin. Large histopathology slides were used to whole-mount entire sections for digitization. The correct sequence was maintained to assist in subsequent reconstruction. Using Photoshop (Adobe Systems Incorporated, San Jose, CA, U.S.A.), contours were placed on the photographic images to represent the external borders of the section and the extent of macroscopic disease. Sections were stacked in sequence and manually oriented in Photoshop. The macroscopic tumour contours were then transferred to MATLAB (The Mathworks, Natick, MA, U.S.A.) and stacked, producing 3D surface renderings of the resected specimen and embedded gross tumour. To evaluate the microscopic extent of disease, customized "tile-based" and commercial confocal panoramic laser scanning (TISSUEscope: Biomedical Photometrics, Waterloo, ON) systems were used to generate digital images of whole-mount histopathology sections. Using the digital whole-mount images and imaging software, we contoured the gross and microscopic extent of disease. Two methods of registering pathology and imaging were used. First, selected pet and ct images were transferred into Photoshop, where they were contoured, stacked, and reconstructed. After importing the pathology and the imaging contours to MATLAB, the contours were reconstructed, manually rotated, and rigidly registered. In the second method, MATLAB tumour renderings were exported to a software platform for manual registration with the original pet and ct images in multiple planes. Data from this software platform were then exported to the Pinnacle radiation treatment planning system in DICOM (Digital Imaging and Communications in Medicine) format. CONCLUSIONS: There is no one definitive method for 3D volumetric RPC in nsclc. An innovative approach to the 3D reconstruction of resected nsclc specimens incorporates agar embedding of the specimen and whole-mount digital histopathology. The reconstructions can be rigidly and manually registered to imaging modalities such as ct and pet and exported to a radiation treatment planning system.

Sentinel lymph nodes in early stage cervical cancer.

OBJECTIVES: Lymph node status is the most important prognostic factor in cervical cancer. Sentinel lymph node (SLN) procedures have been purported to reduce peri- and postoperative morbidity and operative time. METHODS: All patients with surgically managed clinical FIGO stage IA/B1 cervical cancer underwent SLN followed by pelvic lymphadenectomy with technetium+/-lymphazurin from April 2004 to April 2006. 0.1-0.2 mci of filtered sulfur colloid technetium was injected submucosally into 4 quadrants of the exocervix. Lymphazurin (4cc) was only used if technetium was unsuccessful in identifying bilateral sentinel lymph nodes. Serial microsections at 5 microm intervals were performed and stained intraoperatively. Complete pelvic node dissections were performed in all patients. RESULTS: Forty-two patients underwent SLN, prior to full pelvic lymphadenectomy. Thirty-nine patients were included for the purposes of this study. The incidence in detecting at least one sentinel node was 98% per patient, and 85% per side. Identification of bilateral sentinel lymph nodes was successful in 28 cases (72%). The median number of SLN/side was 2. Three patients were found to have metastatic tumor to lymph nodes. No false negatives were identified. No adverse effects were noted. CONCLUSIONS: SLN biopsy in cervical cancer is feasible to do, with a low false negative rate. We believe SLN should be evaluated per side and not per patient, that a pelvic lymphadenectomy is otherwise required. By following this protocol, the false negative rate can be minimized. The combined reported FN rate in the literature is 1.8%. If our definition is applied, the majority of reported false negative SLN is not actual false negatives.

Tsg101 control of human immunodeficiency virus type 1 Gag trafficking and release.

The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55(gag), contains a proline-rich motif (PTAP) called the "late domain" in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of gag-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding.

Observer variation in contouring gross tumor volume in patients with poorly defined non-small-cell lung tumors on CT: the impact of 18FDG-hybrid PET fusion.

PURPOSE: To quantify interobserver variation in gross tumor volume (GTV) localization using CT images for patients with non-small-cell lung carcinoma and poorly defined tumors on CT and to determine whether variability would be reduced if coregistered 2-[18F]fluoro-2-deoxy-d-glucose (FDG)-hybrid positron emission tomography (PET) with CT images were used. METHODS AND MATERIALS: Prospectively, 30 patients with non-small-cell lung carcinoma had CT and FDG-hybrid PET examinations in radiation treatment position on the same day. Images were coregistered using eight fiducial markers. Guidelines were established for contouring GTVs. Three radiation oncologists performed localization independently. The coefficient of variation was used to assess interobserver variability. RESULTS: The size of the GTV defined showed great variation among observers. The mean ratios of largest to smallest GTV were 2.31 and 1.56 for CT only and for CT/FDG coregistered data, respectively. The addition of PET reduced this ratio in 23 of 30 cases and increased it in 7. The mean coefficient of variation for GTV based on the combined modalities was significantly smaller (p < 0.01) than that for CT data only. CONCLUSIONS: High observer variability in CT-based definition of the GTV can occur. A more consistent definition of the GTV can often be obtained if coregistered FDG-hybrid PET images are used.

Combined large cell neuroendocrine carcinoma and spindle cell carcinoma of the lung.

We report a unique case of a combined pulmonary large-cell neuroendocrine carcinoma and spindle-cell carcinoma. The patient was a 54-year-old female smoker who presented with a 4-month history of increased left-sided chest pain and exertional dyspnea. The left upper lobectomy specimen revealed an 8.0-cm mass with central necrosis. Microscopically, the epithelial areas were composed of well-defined nests of large cells with peripheral palisading expressing low-molecular-weight keratin, synaptophysin, chromogranin, and neuron-specific enolase. The spindle-cell component consisted of pleomorphic cells arranged in fibrosarcoma and malignant fibrous histiocytoma-like patterns. These spindle cells were positive for low-molecular-weight keratin and vimentin with focal expression of CD68 and muscle-specific actin. Electron microscopy in the spindle-cell areas showed cell junctions and numerous tonofilaments, indicative of epithelial differentiation. The tumor behaved aggressively and the patient died with extensive metastases 4 months after surgery. The combination of neuroendocrine malignancies and spindle-cell carcinomas appears to be uncommon in the lung. Previous reports have described this association in single case reports of anaplastic small-cell carcinoma and atypical carcinoid, but not in large-cell neuroendocrine carcinoma.

Early experience in intermediate-risk penile cancer with sentinel node identification using the gamma probe.

OBJECTIVES: To report the initial experience with sentinel node identification using the gamma probe in patients with intermediate-risk penile cancer (T2NXM0, or T1 with intermediate or high-grade disease) and impalpable groin nodes. METHODS: Technetium-99m-labeled sulfur colloid was injected at the site of primary penile carcinoma 1 hour before surgery. The sentinel lymph nodes were located using the gamma probe and excised through a 3-cm inguinal incision. A full groin dissection was performed only in cases in which frozen section of the node demonstrated metastasis. RESULTS: Nine sentinel nodes were identified by the gamma probe and excised in 5 men. In 3 patients, the sentinel nodes were negative bilaterally. In 2 patients, the sentinel node, although grossly normal, showed a single focus of metastasis by frozen section analysis. In both of these patients, a full groin dissection was carried out and revealed no other nodal metastases. All 5 remained free of recurrence (median follow-up 18 months, range 16 to 23). CONCLUSIONS: In patients with microscopic involvement of a single lymph node only (confirmed by full groin dissection), gamma probe identification was 100% accurate. None of the patients with negative sentinel nodes had a recurrence. Biopsy of the sentinel nodes using the gamma probe can predict the presence or absence of inguinal node metastasis in patients with intermediate-risk penile cancer, sparing many patients the long-term morbidity of a full groin dissection. These initial results suggest further study is warranted.

HIV-1 capsid protein forms spherical (immature-like) and tubular (mature-like) particles in vitro: structure switching by pH-induced conformational changes.

The viral genome and replicative enzymes of the human immunodeficiency virus are encased in a shell consisting of assembled mature capsid protein (CA). The core shell is a stable, effective protective barrier, but is also poised for dissolution on cue to allow transmission of the viral genome into its new host. In this study, static light scattering (SLS) and dynamic light scattering (DLS) were used to examine the entire range of the CA protein response to an environmental cue (pH). The CA protein assembled tubular structures as previously reported but also was capable of assembling spheres, depending on the pH of the protein solution. The switch from formation of one to the other occurred within a very narrow physiological pH range (i.e., pH 7.0 to pH 6.8). Below this range, only dimers were detected. Above this range, the previously described tubular structures were detected. The ability of the CA protein to form a spherical structure that is detectable by DLS but not by electron microscopy indicates that some assemblages are inherently sensitive to perturbation. The dimers in equilibrium with these assemblages exhibited distinct conformations: Dimers in equilibrium with the spherical form exhibited a compact conformation. Dimers in equilibrium with the rod-like form had an extended conformation. Thus, the CA protein possesses the inherent ability to form metastable structures, the morphology of which is regulated by an environmentally-sensitive molecular switch. Such metastable structures may exist as transient intermediates during the assembly and/or disassembly of the virus core.

Structural consequences of cyclophilin A binding on maturational refolding in human immunodeficiency virus type 1 capsid protein.

While several cellular proteins are incorporated in the human immunodeficiency virus type 1 virion, cyclophilin (CyP) A is the only one whose absence has been demonstrated to impair infectivity. Incorporation of the cytosolic protein results from interaction with a highly exposed Pro-rich loop in the N-terminal region of the capsid (CA) domain of the precursor polyprotein, Pr55(Gag). Even when prevented from interacting with CyP A, Pr55Gag still forms particles that proceed to mature into morphologically wild-type virions, suggesting that CyP A influences a postassembly event. The nature of this CyP A influence has yet to be elucidated. Here, we show that while CyP A binds both Gag and mature CA proteins, the two binding interactions are actually different. Tryptophan 121 (W121) in CyP A distinguished the two proteins: a phenylalanine substitution (W121F) impaired binding of mature CA protein but not of Gag. This indicates the occurrence of a maturation-dependent switch in the conformation of the Pro-rich loop. A structural consequence of Gag binding to CyP A was to block this maturational refolding, resulting in a 24-kDa CA protein retaining the immature Pro-rich loop conformation. Using trypsin as a structure probe, we demonstrate that the conformation of the C-terminal region in mature CA is also a product of maturational refolding. Binding to wild-type CyP A altered this conformation, as indicated by a reduction in the accessibility of Cys residue(s) in the region to chemical modification. Hence, the end result of binding to CyP A, whether the Pro-rich loop is in the context of Gag or mature CA protein, is a structurally modified mature CA protein. The postassembly role of CyP A may be mediated through these modified mature CA proteins.

Spatial and temporal registration of CT and SPECT images: development and validation of a technique for in vivo three-dimensional semiquantitative analysis of bone.

UNLABELLED: The combined use of postoperative 3-dimensional CT and SPECT imaging provides a means of relating anatomy and physiology for the semiquantitative in vivo analysis of bone. This study focuses on the development and validation of a technique that accomplishes this through the registration of SPECT data to a 3-dimensional volume of interest (VOI) interactively defined on CT images. METHODS: Five human cadaver heads served as anthropomorphic models for all experiments. Four cranial defects were created in each specimen with inlay and onlay split-skull bone grafts reconstructed to skull and malar recipient sites. To acquire all images, each specimen was landmarked with 1.6-mm ball bearings and CT scanned. Bone surfaces were coated with 99mTc-doped paint. The locations of the ball bearings were marked with paint doped with 111In. Separate SPECT scans were acquired using the energy windows of 99mTc and 111In. RESULTS: Serial SPECT images aligned with an average root-mean-square (RMS) error of 3.8 mm (i.e., <1 pixel). CT-to-SPECT volume matching aligned with an RMS error of 7.8 mm. Total counts in CT-defined VOIs applied to SPECT data showed a strong linear correlation (r2 = 0.86) with true counts obtained from a dose calibrator. CONCLUSION: The capability of this multimodality registration technique to anatomically localize and quantify radiotracer uptake is sufficiently accurate to warrant further assessment in an in vivo trial.

Cytokine effects on glutamate uptake by human astrocytes.

Glutamate uptake by astrocytes has been postulated to play a neuroprotective role during brain inflammation. Using primary human fetal astrocyte cultures, we investigated the influence of selected cytokines on glutamate uptake activity. Interleukin (IL)-1beta and tumor necrosis factor-alpha dose-dependently inhibited astrocyte glutamate uptake, whereas interferon (IFN)-gamma alone stimulated this activity. The nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine, blocked IL-1beta-mediated inhibition of glutamate uptake, suggesting involvement of nitric oxide in the effect of IL-1beta. IL-1 receptor antagonist protein totally reversed the inhibitory effect of cytokines, suggesting a critical role of IL-1beta. The anti-inflammatory cytokine IFN-beta blocked cytokine (IL-1beta plus IFN-gamma)-induced inhibition of glutamate uptake with a corresponding reduction in nitric oxide generation. Taken together, these findings suggest that proinflammatory cytokines inhibit astrocyte glutamate uptake by a mechanism involving nitric oxide, and that IFN-beta may exert a therapeutically beneficial effect by blocking cytokine-induced nitric oxide production in inflammatory diseases of the brain.

Preparation of polyetherol-appended sulfur porphyrazines and investigations of peripheral metal ion binding in polar solvents.

We describe the complete synthesis and characterization of a new family of peripherally functionalized porphyrazines (pz's) with four, three, or two (in a trans conformation) bis[thioethoxy(ethoxy)ethanol] moieties appended at the pyrroles. These "polyetherol" groups serve as weak exocyclic binding sites for a number of metal ions and also provide solubility of the pz's in low molecular-weight alcohols and water. Electronic spectra of the modified porphyrazines exhibit distinct changes in the visible region (both absorbance and fluorescence) in response to treatment with Ag+, Pb2+, Cd2+, Cs+, and Ni2+ in solution. Such properties make these compounds intriguing candidates for incorporation into the transducer layers in optically based chemical sensors.