Abstract Heparin-induced thrombocytopenia (HIT) is characterized by mild thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human (h) platelet factor 4 (PF4) complexed with various polyanions. While non-heparin anticoagulants and intravenous immunoglobulin (IVIG) are the mainstay of care, bleeding may develop, and risk of new thromboembolic events remain. We had described a mouse IgGκ2b antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4:polyanion complexes. KKO, like HIT IgGs, activates platelets through FcγRIIA and induces complement activation. We now asked whether Fc-modified KKO can be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). DGKKO bound to PF4-polyanion complexes, and blocked FcγRIIA-dependent activation of PF4 treated platelets by KKO, 5B9 (another HIT-like monoclonal antibody), and isolated IgGs from HIT patients. DGKKO also decreased complement activation and deposition of C3c on platelets. Injection of DGKKO into “HIT mice” lacking mouse PF4, but transgenic for hPF4 and FcγRIIA, prevented and reversed thrombocytopenia when injected before or after KKO, 5B9 or HIT IgG, respectively, in a microfluidic system. DGKKO reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis by IgG from a patient with the HIT-related disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT. Key Points Deglycosylated (DG) KKO can reverse thrombocytopenia in a HIT murine model. DGKKO can prevent/reverse thrombosis in vitro and in a HIT murine model.
Synovial inflammation, angiogenesis and joint degradation are the hallmarks of inflammatory arthritis progression. Angiostatic targeting is an extensively studied potential therapeutic option for inflammatory arthritis. Studies have confirmed that surface-active phospholipids (SAPLs), predominantly phosphatidylcholines (PCs), are responsible for the lubricating properties of lubricin in joints. Paclitaxel, a potent antineoplastic agent in cancer chemotherapy, has been shown to inhibit several processes associated with arthritis development such as angiogenesis, neutrophil activation and collagenase expression but is limited by systemic toxicity. This study was aimed at designing a surface-active phospholipid mimetic nanocarrier system and assessing its efficacy for intra-articular delivery of paclitaxel in rat joints. Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes were prepared using a thin-film hydration method and characterized for size, morphology, drug encapsulation and in vitro release. DPPC liposomes of a size of 311 ± 57 nm and 92 ± 0.6% paclitaxel encapsulation were developed. In vitro release studies showed a short initial burst phase and a sustained release profile with a cumulative release of 18 ± 0.36% of the drug by 60 h in phosphate-buffered saline (PBS). The efficacy of the intra-articular formulation was evaluated in antigen-induced arthritic rat models and compared with direct injections of paclitaxel. After a 28-day period, intra-articular paclitaxel delivered in liposomes led to a significant improvement in gait scores and synovial inflammation in rats compared to the control, as seen in histopathology studies. Reduction in inflammation in the experimental group was confirmed by evaluating TNFα levels in serum samples. This study suggests feasibility of using surface-active phospholipid based carriers for local, intra-articular therapy of paclitaxel in arthritis.
There is a strong association between depression and memory impairment. The present study aims to assess the nootropic activity of duloxetine and piracetam combination. Male Swiss Albino mice were divided randomly into 4 groups. Treatment of normal saline (10 ml/kg), duloxetine (10 mg/kg), piracetam (100 mg/kg), and duloxetine (5 mg/kg) plus piracetam (50 mg/kg) were given through intra-peritoneal route to group I-IV, respectively. Transfer latency in elevated plus maze (EPM) and time spent in target quadrant in Morris water maze (MWM) were recorded. Estimation of brain monoamines in hippocampus, cerebral cortex, and whole brain were done using HPLC with fluorescence detector. Piracetam treated group showed significant decrease in transfer latency in EPM and increase in time spent in target quadrant recorded in MWM. Combination treated group failed to produce statistically significant nootropic effect in both EPM and MWM. Combination treated group failed to increase brain monoamine levels when compared against duloxetine and piracetam treated groups, separately. But there was exception of significant increase in norepinephrine levels in hippocampi when compared against duloxetine treated group. Results indicate no cognitive benefits with piracetam plus duloxetine combination. These findings can be further probed with the aim of understanding the interaction between duloxetine and piracetam as a future endeavor.
Platelets are highly reactive fragments of megakaryocytes that play a fundamental role in thrombosis and hemostasis. Predictably, all conventional anti-platelet therapies elicit bleeding, raising the question whether the thrombotic activity of platelets can be targeted separately. In this study, we describe a novel approach of inhibiting platelet activation through the use of bispecific single-chain variable fragments (bi-scFvs), termed cis-acting platelet receptor inhibitors (CAPRIs) that harness the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing co-inhibitory receptor G6b-B (G6B) to suppress immunoreceptor tyrosine-based (ITAM)-containing receptor-mediated platelet activation. CAPRI-mediated hetero-clustering of G6B with either the ITAM-containing GPVI-FcR γ-chain complex or FcγRIIA (CD32A) inhibited collagen- or immune complex-induced platelet aggregation. G6B-GPVI CAPRIs strongly and specifically inhibited thrombus formation on collagen under arterial shear, whereas G6B-CD32A CAPRI strongly and specifically inhibited thrombus formation to heparin-induced thrombocytopenia, vaccine-induced thrombotic thrombocytopenia and antiphospholipid syndrome complexes on Von Willebrand Factor-coated surfaces and photochemical-injured endothelial cells under arterial shear. Our findings provide proof-of-concept that CAPRIs are highly effective at inhibiting ITAM receptor-mediated platelet activation, laying the foundation for a novel family of anti-thrombotic therapeutics with potentially improved efficacy and fewer bleeding outcomes compared with current anti-platelet therapies.
Colon carcinoma is the third largest cause of death in United States. 5-Fluorouracil (FUra)-based chemotherapy has contributed significantly in improving the prognosis of this disease. Earlier studies performed in our laboratories have established the significant advantage of using FUra/ Leucovorin (LV) + IFN-gamma in obtaining selective and synergistic therapeutic action. In the current study, five different colon carcinoma cell lines (RKO, HT29, GC3, HCT8 and HCT116) were treated with different combinations of FUra/LV and IFN-gamma. A folate-based thymidylate synthase inhibitor (BGC9331) was also utilized in HCT116 cells that induced DNA damage in contrast to FUra/LV, which induced RNA mediated damage. All cell lines were treated for 24 hr and total RNA was isolated after harvesting the cells. All reactions were carried out in triplicate. Total RNA isolated from treated and control cell lines was used for carrying out Illumina cDNA microarray analysis (RF-8). The results obtained were analyzed using Beadstudio software available from Illumina (Gene Expression Analysis module). The data obtained were initially analyzed using Cluster analysis dendrograms to confirm the groups and subgroups of cell lines with different treatment designs. The data were validated using all four clustering metrics: Correlation, Absolute Correlation, Manhattan and Euclidian. Any outlier groups were eliminated from further analysis. Scatter analysis was used to determine genes that were significantly altered in their expression levels in response to a particular drug treatment or in combination. Bar plot analysis was conducted to visualize the change in expression level of a particular gene in response to different treatments in different cell lines. Of specific interest was that a significant reduction in levels of the thymidylate synthase gene expression was detected in response to IFN-gamma. The difference was most pronounced in RKO cells. Similarly other genes of interest are currently being studied individually including those known to be involved in the synergistic interaction between FUra, LV and IFN-gamma. We are also elucidating the differential expression of sets of genes known to be involved in important signaling pathways including Fas, Wnt and Hedgehog as well as elucidating gene signature(s) that will differentiate DNA damage (toxic to colon cancer cells) from RNA damage (that initiates GI toxicity). It is anticipated that these studies will improve our understanding of molecular targets for FUra-based therapy in colon cancer and will aid in elucidating novel drug targets that will be more effective and selective for colon cancer therapy. Supported by NCI awards CA32613 and CA108929 Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2436.
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