Endothelial ERG programs neutrophil transcriptome for sustained anti-inflammatory vascular niche.

Neutrophils (PMNs) reside as a marginated pool within the vasculature, ready for deployment during infection. However, how endothelial cells (ECs) control PMN extravasation and activation to strengthen tissue homeostasis remains ill-defined. Here, we found that the vascular ETS-related gene (ERG) is a generalized mechanism regulating PMN activity in preclinical tissue injury models and human patients. We show that ERG loss in ECs rewired PMN-transcriptome, enriched for genes associated with the CXCR2-CXCR4 signaling. Rewired PMNs compromise mice survival after pneumonia and induced lung vascular inflammatory injury following adoptive transfer into naïve mice, indicating their longevity and inflammatory activity memory. Mechanistically, EC-ERG restricted PMN extravasation and activation by upregulating the deubiquitinase A20 and downregulating the NFκB-IL8 cascade. Rescuing A20 in EC-Erg -/- endothelium or suppressing PMN-CXCR2 signaling rescued EC control of PMN activation. Findings deepen our understanding of EC control of PMN-mediated inflammation, offering potential avenues for targeting various inflammatory diseases. Highlights: ERG regulates trans-endothelial neutrophil (PMN) extravasation, retention, and activationLoss of endothelial (EC) ERG rewires PMN-transcriptomeAdopted transfer of rewired PMNs causes inflammation in a naïve mouse ERG transcribes A20 and suppresses CXCR2 function to inactivate PMNs. In brief/blurb: The authors investigated how vascular endothelial cells (EC) control polymorphonuclear neutrophil (PMN) extravasation, retention, and activation to strengthen tissue homeostasis. They showed that EC-ERG controls PMN transcriptome into an anti-adhesive and anti-inflammatory lineage by synthesizing A20 and suppressing PMNs-CXCR2 signaling, defining EC-ERG as a target for preventing neutrophilic inflammatory injury.

RGS2 is an innate immune checkpoint for TLR4 and Gαq-mediated IFNγ generation and lung injury.

IFNγ, a type II interferon secreted by immune cells, augments tissue responses to injury following pathogenic infections leading to lethal acute lung injury (ALI). Alveolar macrophages (AM) abundantly express Toll-like receptor-4 and represent the primary cell type of the innate immune system in the lungs. A fundamental question remains whether AM generation of IFNg leads to uncontrolled innate response and perpetuated lung injury. LPS induced a sustained increase in IFNg levels and unresolvable inflammatory lung injury in the mice lacking RGS2 but not in RGS2 null chimeric mice receiving WT bone marrow or receiving the RGS2 gene in AM. Thus, indicating RGS2 serves as a gatekeeper of IFNg levels in AM and thereby lung's innate immune response. RGS2 functioned by forming a complex with TLR4 shielding Gaq from inducing IFNg generation and AM inflammatory signaling. Thus, inhibition of Gaq blocked IFNg generation and subverted AM transcriptome from being inflammatory to reparative type in RGS2 null mice, resolving lung injury. Highlights: RGS2 levels are inversely correlated with IFNγ in ARDS patient's AM.RGS2 in alveolar macrophages regulate the inflammatory lung injury.During pathogenic insult RGS2 functioned by forming a complex with TLR4 shielding Gαq from inducing IFNγ generation and AM inflammatory signaling. eToc Blurb: Authors demonstrate an essential role of RGS2 in macrophages in airspace to promoting anti-inflammatory function of alveolar macrophages in lung injury. The authors provided new insight into the dynamic control of innate immune response by Gαq and RGS2 axis to prevent ALI.

Protease-activated receptor 2 promotes clearance of Pseudomonas aeruginosa infection by inducing cAMP-Rac1 signaling in alveolar macrophages.

Efficient phagocytosis of pathogens by the innate immune system during infectious injury is vital for restoring tissue integrity. Impaired phagocytosis, such as in the case of infection with Pseudomonas aeruginosa, a broad-spectrum antibiotic-resistant Gram-negative bacterium, can lead to a life threatening lung disorder, acute lung injury (ALI). Evidence indicates that loss of protease-activated receptor 2 (PAR2) impaired Pseudomonas aeruginosa clearance leading to non-resolvable ALI, but the mechanism remains unclear. Here, we focused on the alveolar macrophages (AMs), the predominant population of lung-resident macrophages involved in sensing bacteria, to understand their role in PAR2-mediated phagocytosis of Pseudomonas aeruginosa. We found that upon binding Pseudomonas aeruginosa, PAR2-expressing but not PAR2-null AMs had increased cAMP levels, which activated Rac1 through protein kinase A. Activated Rac1 increased actin-rich protrusions to augment the phagocytosis of Pseudomonas aeruginosa. Administration of liposomes containing constitutively active Rac1 into PAR2-null mice lungs rescued phagocytosis and enhanced the survival of PAR2-null mice from pneumonia. These studies showed that PAR2 drives the cAMP-Rac1 signaling cascade that activates Pseudomonas aeruginosa phagocytosis in AMs, thereby preventing death from bacterial pneumonia.

A Co(III) Complex of 1-Amino-4-hydroxy-9,10-anthraquinone Exhibits Apoptotic Action against MCF-7 Human Breast Cancer Cells.

A Co(III) complex of 1-amino-4-hydroxy-9,10-anthraquinone (QH) (Scheme-1) having the molecular formula CoQ3 (Scheme-2) was prepared and characterized by elemental analysis, FTIR spectroscopy, UV-vis spectroscopy, fluorescence spectroscopy, and mass spectrometry. In the absence of a single crystal, the energy-optimized molecular structure of CoQ3 was determined by employing computational methods that was validated using spectroscopic evidences, elemental analysis, and mass spectrometry data. The electrochemical properties of the complex were analyzed using cyclic voltammetry and indicate a substantial modification of the electrochemical properties of the parent amino-hydroxy-9,10-anthraquinone. CoQ3 was thereafter tested on MCF-7 human breast cancer cells. The IC50 value for a 24 h incubation was found to be (95 ± 0.05) µg/mL. The study showed that such cancer cells underwent both early and late apoptosis following the interaction with CoQ3.

Leishmania donovani infection induce Extracellular signal-regulated kinase ½ (ERK½) mediated lipid droplet generation in macrophages.

Recently unfolded mechanisms showed lipid droplet helps in pathogen survival and paralyzes host immune response. In the present study, we showed the extent of lipid droplet(LD) generation in Leishmania donovani infection, the signaling involved, and their function concerning pathogenicity. RAW 264.7 and J774A.1 cells were used to infect with L. donovani and then flow cytometry and confocal microscopy were used to detect lipid droplet generation and subsequent assays. In this study, we showed that L. donovani AG83 (AG83/MHOM/1983) triggers lipid droplet formation in macrophages in a time-dependent manner. We provide novel insight into the signaling molecules which is responsible for LD accumulation. Interestingly, LPG deficient attenuated Leishmania strain UR6 (UR6/MHOM/1978) failed to fuel LD generation. But inhibition of phagosome maturation drastically stimulates LD accumulation in UR6 infected MΦs. Aspirin treatment in AG83 infected MΦs does not only lower LD load but also favors phagolysosome biogenesis and corrects cytokine balance. Employing strategies to circumvent halt in phagosome maturation using drugs that manipulate lipid droplet generation could be used as a therapeutic tool to resist parasite growth in the early hour of infection.

Evidence for reprogramming of monocytes into reparative alveolar macrophages in vivo by targeting PDE4b.

Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMϕ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMϕ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMϕ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMϕ and regulation of mobilization of the reparative AMϕ-pool. RNA-seq analysis of flow-sorted Mϕ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for the transcription of PDE4b in Mϕ. Inhibition of PDE4 activity at the time of peak injury, using intratracheal rolipram, increased cAMP levels, augmented the reparative AMϕ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMϕ. Interestingly, adoptive transfer of rolipram-educated AMϕ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMϕ generation from monocytes for lung repair.

Macrophage TLR4 and PAR2 Signaling: Role in Regulating Vascular Inflammatory Injury and Repair.

Macrophages play a central role in dictating the tissue response to infection and orchestrating subsequent repair of the damage. In this context, macrophages residing in the lungs continuously sense and discriminate among a wide range of insults to initiate the immune responses important to host-defense. Inflammatory tissue injury also leads to activation of proteases, and thereby the coagulation pathway, to optimize injury and repair post-infection. However, long-lasting inflammatory triggers from macrophages can impair the lung's ability to recover from severe injury, leading to increased lung vascular permeability and neutrophilic injury, hallmarks of Acute Lung Injury (ALI). In this review, we discuss the roles of toll-like receptor 4 (TLR4) and protease activating receptor 2 (PAR2) expressed on the macrophage cell-surface in regulating lung vascular inflammatory signaling.

Inhibition of TGF-ß induced lipid droplets switches M2 macrophages to M1 phenotype.

Lipid droplets (LD) are newly characterized dynamic cytoplasmic organelle which is the storehouse of different immunosuppressive cytokines and enzymes like cyclooxygenase and lipoxygenase. Tumors are known to modulate the immune system by immune-editing the microenvironment. Immuno-editing comprises of three steps namely cancer immune-surveillance, tumor dormancy and finally escape leading to tumor development. The latency of the tumor microenvironment is greatly contributed by the M2 polarized macrophages and TGF-ß is a prime culprit. Modulating M2 macrophages to M1 can be a strategy against tumor progression. We found that tumor-conditioned medium or recombinant TGF-ß was efficient to induce LD formation in Raw264.7 cells and the inhibition of LD was associated with the switch of M2 to M1 phenotype involving MEK1/2 axis. Signature molecules of M2 polarized macrophages like CD206 were also downregulated while co-stimulatory molecules like CD80, CD86 were up-regulated along with enhanced surface expression of MHCII when these macrophages were subjected to C75 treatment to reduce the LD formation. The level of pro-inflammatory cytokine, as well as ROS and NO generation, were also increased when TGF-ß treated macrophages were subjected to C75 treatment. This study is probably the first report of this kind and can be used in the future in cancer treatment.

Bromelain with peroxidase from pineapple are more potent to target leukemia growth inhibition - A comparison with only bromelain.

The natural anti-cancer agent bromelain is found to be beneficial for either single or multi-targeted therapy in gastric and skin carcinoma, by inhibiting cancer cell growth. Importantly, the presence of peroxidase enhances its biological efficiency. We have now evaluated a panel of cancer cell lines with bromelain in presence or absence of peroxidase to identify that the combination has higher apoptosis inducing potential in all those cell lines. Bromelain plus peroxidase (BM-PR) inhibited acute myeloid (K562) cell proliferation and altered the morphological features. Incidence of apoptosis was established by using annexin V exposure and this was confirmed that the cell cycle was arrested at G0/G1 phase in a concentration-dependent manner. BM-PR increased the intracellular ROS level and altered the mitochondrial membrane potential, as detected using dichlorofluores cin diacetate (DCFDA). It also regulated the expression of apoptosis-related proteins like Bax, Bcl2, caspase-3 and cytochrome besides causing up-regulation of p53 as determined by western blot analysis. These results suggest that BM-PR from pineapple induces apoptosis better than only bromelain in acute myeloid leukemia cells possibly via mitochondria dependent pathway.

Pyridine-antipyrine appended indole derivative for selective recognition of Fe3+: Concentration dependent coloration.

Combination of pyridine, antipyrine and indole in a single molecule (L2) allows selective recognition of Fe3+ colorimetrically in CH3CN. The structure of L2 is confirmed from single crystal X-ray diffraction analysis. The probe displays two different visible bands at 541nm and 715nm in the presence of Fe3+, associated with two different colors, viz. green and pink-violet allowing determination of unknown Fe3+ concentration. Interestingly, removal of 2-picolyl group from indole N-center of L2 generates L3 that behaves similarly at low Fe3+ concentration (>0 to 1.1mM) but differently at higher Fe3+ concentration (>1.1mM), indicating involvement of pyridyl-N donor towards Fe3+, and hence different coordination environment around Fe3+ at higher concentration.

Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages.

Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain pre-exposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in future.

Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway.

BACKGROUND/AIMS: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. METHODS: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. RESULTS: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. CONCLUSION: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy.

Antineoplastic impact of leishmanial sphingolipid in tumour growth with regulation of angiogenic event and inflammatory response.

Very often conventional therapy, i.e. chemotherapeutic treatment, develops resistance in cancer cells and fails to be effective against disease states. An alternative strategy or a new entity may resolve the problem. Interestingly, the microbial world has begun to be explored in medicinal research as a potential new source to deliver bio-active molecules such as sphingolipids for efficacious cancer treatment. A sphingolipid of microbial origin, especially from Leishmania donovani (LSPL), is a novel entity which may exert anti-cancer activity by regulating cellular growth. The present study reveals that among a range of cancer cells evaluated, LSPL-1 (a component of LSPL) reduces cell viability, annexin exposures and arrests cell cycle in B16F10 cells in a concentration and time dependent manner. Flowcytometric analysis showed that it alters mitochondrial membrane potential and generates a number of ROS positive melanoma cells. It activates p53 at serine anchor region via up-regulation of p21 subunit along with PUMA and NOXA. It also exerts activity in vivo by reducing tumor micro vessel and mitotic index while simultaneously improving the survival rate. The inflammatory responses including elevated level of cytokine-chemokine and increased expression of PCNA and F480 are subdued by LSPL-1 treatment in tumour bearing mice. Besides, it reduces the metastatic outburst of angiogenic factors like VEGF, Ang-2, and CD34 through the involvement of several growth promoting factors. These findings indicate that LSPL-1 may be explored as a potent entity against melanoma growth and the associated angiogenic promotion.

Lipid from infective L. donovani regulates acute myeloid cell growth via mitochondria dependent MAPK pathway.

The microbial source, which includes live, attenuated, or genetically modified microbes or their cellular component(s) or metabolites, has gained increasing significance for therapeutic intervention against several pathophysiological conditions of disease including leukemia, which remains an incurable disease till now despite recent advances in the medical sciences. We therefore took up the present study to explore if the leishmanial lipid (pLLD) isolated from L. donovani can play an anti-neoplastic role in acute myeloid leukemia cells by regulating cellular growth. Indeed pLLD significantly inhibited cell proliferation of four AML cell lines (HL-60, MOLT-4, U937, and K562). Scanning electron microscopy and DNA fragmentation analysis revealed that it significantly induced apoptosis of U937 cells through morphological alteration. Occurrence of apoptosis was checked by using Annexin exposure and this established that the cell cycle was arrested at G0/G1 phase in time-dependent manner. pLLD increased the intracellular ROS with alteration of mitochondrial membrane potential, as detected using DCFDA. It also regulated the expression of apoptosis-related proteins like Bax, Bcl2, Bad and t-Bid besides causing cleavage of PARP as determined by western blot analysis. Treatment of U937 cells with pLLD induced the activation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK)1/2, p38, and caspases 9/3. The results suggest that pLLD induces apoptosis in acute myeloid leukemia cells possibly via increasing intracellular ROS and regulating the MAPK pathway.

Leishmanial lipid affords protection against oxidative stress induced hepatic injury by regulating inflammatory mediators and confining apoptosis progress.

Persistence of liver injury alters the internal milieu, promotes deregulation of inflammatory factors, and leads to dysplastic lesions like fibrosis, cirrhosis to hepatocellular carcinoma. Our previous study revealed that leishmanial lipid (pLLD) exerts potential anti-inflammatory activity in sepsis associated hepatic injury. We now show that pLLD gives protection against chemical induced hepatotoxicity in murine system. The beneficial effect of treatment with pLLD on such hepatic injury in mice was analyzed using different assays including ELISA, FACS, western blot and immunohistochemical analysis. pLLD significantly suppressed serum enzymes and rectified the histopathological alteration to induce the antioxidant level in CCl4 intoxicated liver. Levels of several growth factors including TGF-ß, HGF, and EGF were significantly improved in serum and hepatic tissue with consequent reduction of caspase activities and expressions of Bad, Bax, p53, and NF-κBp65. Moreover, pLLD modulated inflammatory responses by decreasing the production of several cytokines and chemokines, thus preventing the infiltration of immune cells to the damaged area. It accelerated the repair process in liver damage with modulation of signalling cascade via alteration of apoptotic factors. Our experimental approaches suggest that pLLD effectively prevents liver injury mainly through down regulation of oxidative stress and inflammatory response towards anti-apoptotic changes.

Leishmanial sphingolipid induces apoptosis in Sarcoma 180 cancer cells through regulation of tumour growth via angiogenic switchover.

Sphingolipids are membrane and intracellular lipids that typically modulate cellular processes to cause cell death. Exogenous administration of sphingolipids may cause restriction of tumour growth and several alternative strategies are being used to control the cell growth. The microbes, their cellular component(s) or metabolites like DHA, EPA and also FTY720 have been employed as new therapeutic entities to regulate the disease condition. The therapeutic efficacy of lipids from Leishmania donovani in rheumatoid arthritis and also in sepsis condition associated with inflammatory diseases is well established. In this study, we explored the apoptotic effect of LSPL-1 (leishmanial sphingolipid-1) in Sarcoma 180 cells towards the regulation of tumour growth. The study using a panel of cancer cell lines revealed that LSPL-1 induces cell death in Sarcoma 180. The apoptotic changes were assessed by annexin exposure and DNA content analysis using flow cytometry. LSPL-1 appears to activate several pro- and anti-apoptotic molecules through reactive oxygen species (ROS) generation and also caspase activation, as determined by Western blot and ELISA analyses. Simultaneously, it may improve the survival rate of mice bearing tumour induced by Sarcoma 180 cells, with pathological changes. LSPL-1 may also suppress the cancer-associated inflammatory responses with the expression of matrix metalloproteinase having inhibitory role. It may regulate several angiogenic factors including VEGF, Ang-2 and CD34 in angiogenic events generated in Sarcoma 180 cell-induced tumour. These studies underline the significance of anti-neoplastic potential of LSPL-1 through apoptosis induction and abrogation of angiogenic responses in Sarcoma 180 cell-associated tumour.

Lipid isolated from a Leishmania donovani strain reduces Escherichia coli induced sepsis in mice through inhibition of inflammatory responses.

Sepsis is the reflection of systemic immune response that manifests in the sequential inflammatory process in presence of infection. This may occur as a result of gram-negative bacterial sepsis including Escherichia coli infection that gives rise to excessive production of inflammatory mediators and causes severe tissue injuries. We have reported earlier that the lipid of attenuated Leishmania donovani suppresses the inflammatory responses in arthritis patients. Using heat killed E. coli stimulated macrophages, we have now investigated the effect of leishmanial total lipid (LTL) isolated from Leishmania donovani (MHO/IN/1978/UR6) for amelioration of the inflammatory mediators and transcriptional factor with suppression of TLR4-CD14 expression. To evaluate the in vivo effect, E. coli induced murine sepsis model was used focusing on the changes in different parameter(s) of lung injury caused by sepsis, namely, edema, vascular permeability, and pathophysiology, and the status of different cytokine-chemokine(s) and adhesion molecule(s). Due to the effect of LTL, E. coli induced inflammatory cytokine-chemokine(s) levels were significantly reduced in serum and bronchoalveolar lavage fluid simultaneously. LTL also improved the lung injury and suppressed the cell adhesion molecules in lung tissue. These findings indicate that LTL may prove to be a potential anti-inflammatory agent and provide protection against gram-negative bacterial sepsis with pulmonary impairment.

Leishmanial lipid suppresses the bacterial endotoxin-induced inflammatory response with attenuation of tissue injury in sepsis.

The use of live, attenuated, or genetically modified microbes or their cellular component(s) or metabolites has begun to emerge as a potential new approach in medicinal research to deliver biologically active entities. Thus, advancing our knowledge of such microbe-mediated therapy may suggest new avenues for therapeutic intervention in many diseases. We had earlier reported that the total lipid of attenuated Leishmania donovani suppressed the inflammatory responses in rheumatoid arthritis patients. Our present study reveals that the pLLD, isolated from pathogenic L. donovani, decreases the inflammatory level of bacterial endotoxin in stimulated mouse macrophages, as also in the in vivo murine system. It exerts the activity by reducing the level of different mediators, such as cytokine-chemokine(s). It also suppresses the expression of the ubiquitous transcription factor NF-κBp65 in stimulated macrophage cells, improves the endotoxin-associated liver damage, reduces the vascular permeability factors, such as VEGF, and suppresses the expression of cell adhesion molecules, including ICAM-1, VCAM-1, PECAM-1, P-selectin, and E-selectin, in liver of septic mice. These findings indicate that pLLD may prove to be a potential anti-inflammatory agent and protect from endotoxin-induced sepsis in hepatic impairment.

Exploring the anti-inflammatory activity of a novel 2-phenylquinazoline analog with protection against inflammatory injury.

Inflammation is a protective immune response against harmful stimuli whose long time continuation results in host disease. Quinazolinones are nitrogen containing heterocyclic compounds with wide spectrum of biological activities. The anticancer effect of a 3-(arylideneamino)-phenylquinazoline-4(3H)-one derivative was reported earlier. The anti-inflammatory effect of these quinazolinone derivatives has now been examined in endotoxin stimulated macrophages and in different in vivo models of inflammation by measuring the proinflammatory cytokines (TNF-α, IL-1ß and IL-6), mediators NO and NF-κB (by ELISA and western blot), and translocation of the nuclear factor kB (by immunocytochemical analysis). To elucidate the in vivo effect, mice endotoxin model was and the various levels of edema, inflammatory pain and vascular permeability were studied. One of the quinazolinone derivatives showed significant anti-inflammatory activity in stimulated macrophage cells by inhibiting the expression of TNF-α, IL-1ß, IL-6, iNOS, COX-2, p-IκB and NF-κBp65. Significant (P<0.01) improvement was observed in the mortality of endotoxemic mice. The carrageenan and formalin-induced paw edema thicknesses were found to be reduced significantly (P<0.01) along with the reduction of pain, vascular permeability and edema induced by complete Freund's adjuvant (P<0.01). These findings indicate that 3-(arylideneamino)-phenylquinazoline-4(3H)-one derivative as a potential anti-inflammatory agent.

Preparation of 99mTc-labelled conjugates of ouabagenin and their biological evaluation in animal models.

BACKGROUND: Ouabagenin and its 1,19-acetonide were conjugated with nitrilotriacetic acid (NTA) and diethylenetriaminepentaacetic acid (DTPA) through their respective anhydrides. METHODS: The reaction mixtures were exhaustively purified by silica gel column chromatography and preparative high-performance liquid chromatography to furnish the ligands in good purity and moderate yield. These ligands were labelled with 99mTc to produce four chelates in 90-95% yield. Of these chelates the 99mTc-oubagenin-NTA conjugate and the corresponding acetonide exhibited appreciable myocardial uptake with respect to that of other vicinal organs in a guinea-pig model. However, all these 99mTc chelates exhibited poor heart-to-blood ratios, which could be attributed to the absence of a 3beta sugar residue in this molecule. CONCLUSION: The result is in agreement with that previously reported in connection with radioiodinated digoxin and digoxigenin derivatives.