Multivalency of non-peptide integrin αVß3 antagonist slows tumor growth.

Multivalency is a powerful strategy for achieving high-affinity molecular binding of compounds to increase their therapeutic potency or imaging potential. In our study, multivalent non-peptide integrin αvß3 antagonists (IA) were designed for antitumor therapy. Docking and molecular dynamics were employed to explore the binding modes of IA monomer, dimer, and trimer. In silico, one IA unit binds tightly in the active site with similar pose to native ligand RGD and other parts of dimer and trimer contribute extra binding affinities by interacting with residues in vicinity of the original site. In vitro studies demonstrated that increasing valency results in increasing antiproliferative and antiorganizational effects against endothelial cells (HUVECs), and a much weaker effect on melanoma B16F10 cells. The antitumor efficacies of the IA multivalent compounds were evaluated in subcutaneous B16F10 melanoma tumor-bearing mice. At 30 mg/kg dose, the mean masses of tumors harvested 18 days after inoculation were significantly reduced (p<10(-7)) by 36±9%, 49±8%, and 71±7% for the IA monomer, dimer, and trimer groups, relative to control. The importance of multivalency was demonstrated to be highly significant beyond the additive effect of the extra pharmacological sites (p=0.00011). These results suggest that the major target of these anti-αvß3 compounds is the neovasculature rather than the cancer cells, and the success of a multivalent strategy depends on the details of the components and linker. This is the first integrin αvß3 multivalent ligand showing clear enhancement in antitumor effectiveness.

Molecular theranostics: a primer for the imaging professional.

OBJECTIVE: A theranostic system integrates some form of diagnostic testing to determine the presence of a molecular target for which a specific drug is intended. Molecular imaging serves this diagnostic function and provides powerful means for noninvasively detecting disease. We briefly review the paradigms rooted in nuclear medicine and highlight recent advances in this field. We also explore how nanometer-sized complexes, called nanomedicines, present an excellent theranostic platform applicable to both drug discovery and clinical use. CONCLUSION: For imagers, molecular theranostics represents a powerful emerging platform that intimately couples targeted therapeatic entities with noninvasive imaging that yields information on the presence of defined molecular targets before, during, and after cognate therapy.

Synthesis and evaluation of a near-infrared fluorescent non-peptidic bivalent integrin alpha(v)beta(3) antagonist for cancer imaging.

Computer modeling approaches to identify new inhibitors are essentially a very sophisticated and efficient way to design drugs. In this study, a bivalent nonpeptide intergrin alpha(v)beta(3) antagonist (bivalent IA) has been synthesized on the basis of an in silico rational design approach. A near-infrared (NIR) fluorescent imaging probe has been developed from this bivalent compound. In vitro binding assays have shown that the bivalent IA (IC(50) = 0.40 +/- 0.11 nM) exhibited improved integrin alpha(v)beta(3) affinity in comparison with the monovalent IA (IC(50) = 22.33 +/- 4.51 nM), resulting in an over 50-fold improvement in receptor affinity. NIR imaging probe, bivalent-IA-Cy5.5 conjugate, also demonstrated significantly increased binding affinity (IC(50) = 0.13 +/- 0.02 nM). Fluorescence microscopy studies showed integrin-mediated endocytosis of bivalent-IA-Cy5.5 in U87 cells which was effectively blocked by nonfluorescent bivalent IA. We also demonstrated tumor accumulation of this NIR imaging probe in U87 mouse xenografts.

Acoustic fingerprints of dye-labeled protein submicrosphere photoacoustic contrast agents.

Dye-labeled protein microspheres, submicron in size and capable of producing thermoelastically generated ultrasound in response to laser stimulation, are presented as contrast agents for photoacoustic imaging. Incident laser energy absorbed by fluorescein isothiocyanate (FITC)-labeled elastin submicrospheres results in thermoelastically generated sound production. Plotted A-line graphs reveal a distinctive morphology and a greater than two orders of magnitude increase in signal amplitude subsequent to converting FITC elastin into submicrospheres (despite a four orders of magnitude decrease in concentration). Evidence of nonlinearity and enhancement of ultrasound backscatter indicate a potential use in contrast-enhanced harmonic imaging. Photoacoustic and ultrasound imaging of FITC-elastin submicrospheres in a water-filled phantom vessel shows enhanced contrast at low concentration and clear delineation of the phantom vessel wall.

Systems diagnostics: the systems approach to molecular imaging.

OBJECTIVE: Molecular imaging has emerged as a powerful technology that has already changed the practice of modern medicine. During this same period, the monumental genome project has sequenced man's entire genetic content. Now the postgenomic aim is to understand the dynamic interactions of the encoded components and their regulatory mechanisms. CONCLUSION: Molecular imaging is well positioned to play a major role in this massive effort as we move toward a comprehensive paradigm for assessing health and disease.

Pulsed high-intensity focused ultrasound enhances apoptosis and growth inhibition of squamous cell carcinoma xenografts with proteasome inhibitor bortezomib.

PURPOSE: To investigate whether combining pulsed high-intensity focused ultrasound (HIFU) with the chemotherapeutic drug bortezomib could improve antitumor activity against murine squamous cell carcinoma (SCC) tumors. MATERIALS AND METHODS: All experiments were conducted with animal care and use committee approval. Murine SCC cells were implanted subcutaneously in C3H mice. When tumors reached 100 mm(3), mice were randomized to one of three groups for twice weekly intraperitoneal injections of 1.5 mg of bortezomib per kilogram of body weight, a proteasome inhibitor (n = 10); 1.0 mg/kg bortezomib (n = 11); or a control vehicle (n = 12). Within each group, half of the mice received pulsed HIFU exposure to their tumors immediately prior to each injection. The time for tumors to reach 650 mm(3) was compared among groups. Additional tumors were stained with terminal deoxynucledotidyl transferase-mediated dUTP nick end labeling and CD31 to assess apoptotic index and blood vessel density, respectively. RESULTS: Tumors in the control group, pulsed HIFU and control group, and 1.0 mg/kg of bortezomib alone group reached the size end point in 5.2 days +/- 0.8 (standard deviation), 5.3 days +/- 0.8, and 5.6 days +/- 1.1, respectively. However, pulsed HIFU and 1.0 mg/kg bortezomib increased the time to end point to 9.8 days +/- 2.9 (P < .02), not significantly different from the 8.8 days +/- 2.1 in tumors treated with 1.5 mg/kg bortezomib alone (P > .05). Combination therapy was also associated with a significantly higher apoptotic index (P < .05). CONCLUSION: Treatment of tumors with pulsed HIFU lowered the threshold level for efficacy of bortezomib, resulting in significant tumor cytotoxicity and growth inhibition at lower dose levels.

Pulsed high-intensity focused ultrasound enhances uptake of radiolabeled monoclonal antibody to human epidermoid tumor in nude mice.

UNLABELLED: The aim of this study was to determine if pulsed high-intensity focused ultrasound (HIFU) exposures could enhance tumor uptake of (111)In-MX-B3, a murine IgG1kappa monoclonal antibody directed against the Le(y) antigen. METHODS: MX-B3 was labeled with (111)In, purified, and confirmed for its binding to the antigen-positive A431 cell line. Groups of nude mice were inoculated subcutaneously with A431 tumor cells on both hind flanks. A tumor on one flank was treated with pulsed-HIFU; the other tumor was used as an untreated control. Within 10 min after the HIFU exposure, the mice received intravenous (111)In-MX-B3 for imaging and biodistribution studies. Mice were euthanized at 1, 24, 48, and 120 h after injection for biodistribution studies. RESULTS: The HIFU exposure shortened the peak tumor uptake time (24 vs. 48 h for the control) and increased the peak tumor uptake value (38 vs. 25 %ID/g [percentage injected dose per gram] for the control). The HIFU effect on enhancing tumor uptake was greater at earlier times up to 24 h, but the effect was gradually diminished thereafter. The HIFU effect on enhancing tumor uptake was substantiated by nuclear imaging studies. HIFU also increased the uptake of the antibody in surrounding tissues, but the net increase was marginal compared with the increase in tumor uptake. CONCLUSION: This study demonstrates that pulsed-HIFU significantly enhances the delivery of (111)In-MX-B3 in human epidermoid tumors xenografted in nude mice. The results of this pilot study warrant further evaluation of other treatment regimens, such as repeated HIFU exposures for greater delivery enhancement of antibodies labeled with cytotoxic radioisotopes or pulsed-HIFU exposure in addition to a combined therapy of (90)Y-B3 and taxol to enhance the synergistic effect.

Pulsed-high intensity focused ultrasound and low temperature-sensitive liposomes for enhanced targeted drug delivery and antitumor effect.

PURPOSE: To determine if pulsed-high intensity focused ultrasound (HIFU) could effectively serve as a source of hyperthermia with thermosensitive liposomes to enhance delivery and efficacy of doxorubicin in tumors. EXPERIMENTAL DESIGN: Comparisons in vitro and in vivo were carried out between non-thermosensitive liposomes (NTSL) and low temperature-sensitive liposomes (LTSL). Liposomes were incubated in vitro over a range of temperatures and durations, and the amount of doxorubicin released was measured. For in vivo experiments, liposomes and free doxorubicin were injected i.v. in mice followed by pulsed-HIFU exposures in s.c. murine adenocarcinoma tumors at 0 and 24 h after administration. Combinations of the exposures and drug formulations were evaluated for doxorubicin concentration and growth inhibition in the tumors. RESULTS: In vitro incubations simulating the pulsed-HIFU thermal dose (42 degrees C for 2 min) triggered release of 50% of doxorubicin from the LTSLs; however, no detectable release from the NTSLs was observed. Similarly, in vivo experiments showed that pulsed-HIFU exposures combined with the LTSLs resulted in more rapid delivery of doxorubicin as well as significantly higher i.t. concentration when compared with LTSLs alone or NTSLs, with or without exposures. Combining the exposures with the LTSLs also significantly reduced tumor growth compared with all other groups. CONCLUSIONS: Combining low-temperature heat-sensitive liposomes with noninvasive and nondestructive pulsed-HIFU exposures enhanced the delivery of doxorubicin and, consequently, its antitumor effects. This combination therapy could potentially produce viable clinical strategies for improved targeting and delivery of drugs for treatment of cancer and other diseases.

Augmentation of targeted delivery with pulsed high intensity focused ultrasound.

This paper reviews the enhanced delivery of genes, drugs and therapeutics using ultrasound. It begins with a general overview of the field and the various techniques associated with it, including sonophoresis, hyperthermia (with ultrasound), sonoporation, and microbubble assisted transvascular and targeted delivery. Particular attention is then paid to pulsed high intensity focused ultrasound drug delivery without the use of ultrasound contrast agents. Feasibility and mechanistic studies of this technique are described in some detail. Conclusions are then drawn regarding possible mechanisms of this treatment, and to contrast with the better known treatments relying on injection of ultrasound contrast agents.

Pulsed-high intensity focused ultrasound enhanced tPA mediated thrombolysis in a novel in vivo clot model, a pilot study.

INTRODUCTION: Thrombotic disease continues to account for significant morbidity and mortality. Ultrasound energy has been investigated as a potential primary and adjunctive treatment for thrombotic disease. We have previously shown that pulsed-high intensity focused ultrasound (HIFU) enhances thrombolysis induced by tissue plasminogen activator (tPA) in vitro, including describing the non-destructive mechanism by which tPA availability and consequent activity are increased. In this study we aimed to determine if the same effects could be achieved in vivo. MATERIALS AND METHODS: In this study, pulsed-HIFU exposures combined with tPA boluses were compared to treatment with tPA alone, HIFU alone and control in a novel in vivo clot model. Clots were formed in the rabbit marginal ear vein and verified using venography and infrared imaging. The efficacy of thrombolytic treatment was monitored via high resolution ultrasonography for 5 h post-treatment. The cross-sectional area of clots at 4 points along the vein was measured and normalized to the pre-treatment size. RESULTS: At 5 h the complete recanalization of clots treated with pulsed-HIFU and tPA was significantly different from the partial recanalization seen with tPA treatment alone. tPA treatment alone showed a significant decrease in clot versus control, where HIFU was not significantly different than control. Histological analysis of the vessel walls in the treated veins showed no apparent irreversible damage to endothelial cells or extravascular tissue. CONCLUSIONS: This study demonstrates that tPA mediated thrombolysis can be significantly enhanced when combined with non-invasive pulsed-HIFU exposures.

Radiolabeled high affinity peptidomimetic antagonist selectively targets alpha(v)beta(3) receptor-positive tumor in mice.

OBJECTIVES: The aim of this research was to synthesize radiolabeled peptidomimetic integrin alpha(v)beta(3) antagonists that selectively target integrin alpha(v)beta(3) receptor and clear rapidly from the whole body. METHODS: Integrin alpha(v)beta(3) antagonists, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonyl-amino-beta-alanine (IA) and 4-[2-(3,4,5,6-tetrahydro-pyrimidin-2-ylamino)-ethyloxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonylamino-beta-alanine hydrochloride (IAC), a hydrophobic carbamate derivative of IA, were conjugated with 2-p-isothiocyanatobenzyl-DOTA at the amino terminus and labeled with (111)In. The (111)In labeled IA and IAC were subjected to in vitro receptor binding, biodistribution and imaging studies using nude mice bearing the receptor-positive M21 human melanoma xenografts. RESULTS: The (111)In-labeled IA (40%) and -IAC (72%) specifically bound in vitro to alpha(v)beta(3) (0.8 microM) at a molar excess. This receptor binding was completely blocked by a molar excess of cold IA to alpha(v)beta(3). The higher receptor-binding affinity of the (111)In-labeled IAC was reflected in higher tumor uptake and retention: 5.6+/-1.4 and 4.5+/-0.7 %ID/g vs. 3.8+/-0.9 and 2.0+/-0.3 %ID/g for the (111)In-labeled IA at 0.33 and 2 h. The tumor uptakes were inhibited by the co-injection of 200 microg of IA, indicating that the uptake was receptor mediated. These antagonists were excreted primarily via the renal system. The (111)In activity retained in the whole body was quite comparable between the (111)In-labeled IA (24% ID) and the (111)In-labeled IAC (33% ID) at 2 h. The higher peak tumor uptake and longer retention resulted in higher tumor-to-background ratios for the (111)In-labeled IAC at 2 h with 9.7, 2.3, 0.8, 1.9, 7.1, 2.2, 0.9, 3.7 and 9.9 for blood, liver, kidney, lung, heart, stomach, intestine, bone and muscle, respectively. The imaging studies with the (111)In-labeled IAC also clearly visualized the receptor-positive tumor at 4 h. CONCLUSIONS: The (111)In-labeled IAC showed an improve tumor targeting kinetics with rapid accumulation and prolonged retention in the alpha(v)beta(3) receptor-positive tumor. This together with the rapid whole-body clearance pharmacokinetics warrants further studies on this IAC analog for molecular imaging of tumor-induced angiogenic vessels and various malignant human tumors expressing the receptor.

Enhancing tissue permeability with MRI guided preclinical focused ultrasound system in rabbit muscle: From normal tissue to VX2 tumor.

High Intensity Focused Ultrasound (HIFU) is an emerging noninvasive, nonionizing physical energy based modality to ablate solid tumors with high power, or increase local permeability in tissues/tumors in pulsed mode with relatively low power. Compared with traditional ablative HIFU, nondestructive pulsed HIFU (pHIFU) is present in the majority of novel applications recently developed for enhancing the delivery of drugs and genes. Previous studies have demonstrated the capability of pHIFU to change tissue local permeability for enhanced drug delivery in both mouse tumors and mouse muscle. Further study based on bulk tissues in large animals and clinical HIFU system revealed correlation between therapeutic effect and thermal parameters, which was absent in the previous mouse studies. In this study, we further investigated the relation between the therapeutic effect of pHIFU and thermal parameters in bulky normal muscle tissues based on a rabbit model and a preclinical HIFU system. Correlation between therapeutic effect and thermal parameters was confirmed in our study on the same bulk tissues although different HIFU systems were used. Following the study in bulky normal muscle tissues, we further created bulky tumor model with VX2 tumors implanted on both hind limbs of rabbits and investigated the feasibility to enhance tumor permeability in bulky VX2 tumors in a rabbit model using pHIFU technique. A radiolabeled peptidomimetic integrin antagonist, 111In-DOTA-IA, was used following pHIFU treatment in our study to target VX2 tumor and serve as the radiotracer for follow-up single-photon emission computed tomography (SPECT) scanning. The results have shown significantly elevated uptake of 111In-DOTA-IA in the area of VX2 tumors pretreated by pHIFU compared with the control VX2 tumors not being pretreated by pHIFU, and statistical analysis revealed averaged 34.5% enhancement 24h after systematic delivery of 111In-DOTA-IA in VX2 tumors pretreated by pHIFU compared with the control VX2 tumors.

Rhodamine-RCA in vivo labeling guided laser capture microdissection of cancer functional angiogenic vessels in a murine squamous cell carcinoma mouse model.

BACKGROUND: Cancer growth, invasion and metastasis are highly related to tumor-associated neovasculature. The presence and progression of endothelial cells in cancer is chaotic, unorganized, and angiogenic vessels are less functional. Therefore, not all markers appearing on the chaotic endothelial cells are accessible if a drug is given through the vascular route. Identifying endothelial cell markers from functional cancer angiogenic vessels will indicate the accessibility and potential efficacy of vascular targeted therapies. RESULTS: In order to quickly and effectively identify endothelial cell markers on the functional and accessible tumor vessels, we developed a novel technique by which tumor angiogenic vessels are labeled in vivo followed by Laser Capture Microdissection of microscopically isolated endothelial cells for genomic screening. Female C3H mice (N = 5) with established SCCVII tumors were treated with Rhodamine-RCA lectin by tail vein injection, and after fluorescence microscopy showed a successful vasculature staining, LCM was then performed on frozen section tissue using the PixCell II instrument with CapSure HS caps under the Rhodamine filter. By this approach, the fluorescent angiogenic endothelial cells were successfully picked up. As a result, the total RNA concentration increased from an average of 33.4 ng/ul +/- 24.3 (mean +/- S.D.) to 1913.4 ng/ul +/- 164. Relatively pure RNA was retrieved from both endothelial and epithelial cells as indicated by the 260/280 ratios (range 2.22-2.47). RT-PCR and gene electrophoresis successfully detected CD31 and Beta-Actin molecules with minimal Keratin 19 expression, which served as the negative control. CONCLUSION: Our present study demonstrates that in vivo Rhodamine RCA angiogenic vessel labeling provided a practical approach to effectively guide functional endothelial cell isolation by laser capture microdissection with fluorescent microscopy, resulting in high quality RNA and pure samples of endothelial cells pooled for detecting genomic expression.

Delivery of liposomal doxorubicin (Doxil) in a breast cancer tumor model: investigation of potential enhancement by pulsed-high intensity focused ultrasound exposure.

RATIONALE AND OBJECTIVES: To investigate the potential of using pulsed high-intensity focused ultrasound (HIFU) exposures to enhance the delivery, and hence therapeutic effect of liposomal doxorubicin (Doxil) in a murine breast cancer tumor model. MATERIALS AND METHODS: Tumors were grown in the bilateral flanks of mice using a mammary adenocarcinoma cell line. Experiments consisted of exposing one of two tumors to pulsed-HIFU, followed by tail vein injections of Doxil. Tumor growth rates were monitored, and assays carried out for doxorubicin concentration in these tumors as well as in a second (squamous cell carcinoma) tumor model and in muscle. Laser scanning confocal microscopy was used with fluorescent probes to observe both the uptake of polystyrene nanoparticles and dilation of exposed blood vessels. Additional experiments involving histologic analysis and real-time temperature measurements were performed to determine the safety of the exposures. RESULTS: Pulsed-HIFU exposures were shown to be safe, producing no apparent deleterious effects in the tumors. The exposures, however, were not found to enhance the delivery of Doxil, and consequently did not allow for lower doses for obtaining tumor regression. Imaging with a fluorescent dextran showed blood vessels to be dilated as a result of the exposures. Experiments with polystyrene nanoparticles of similar size to the liposomes showed a greater abundance to be present in the treated tumors. CONCLUSION: Although past studies have shown the advantages of pulsed-HIFU exposures for enhancing delivery, this was not observed with the liposomes, apparently because of their inherent ability to preferentially accumulate into tumors on their own. Potential mechanisms for enhanced uptake of non-liposomal nanoparticles are discussed.

Remote spatiotemporally controlled and biologically selective permeabilization of blood-brain barrier.

The blood-brain barrier (BBB), comprised of brain endothelial cells with tight junctions (TJ) between them, regulates the extravasation of molecules and cells into and out of the central nervous system (CNS). Overcoming the difficulty of delivering therapeutic agents to specific regions of the brain presents a major challenge to treatment of a broad range of brain disorders. Current strategies for BBB opening are invasive, not specific, and lack precise control over the site and timing of BBB opening, which may limit their clinical translation. In the present report, we describe a novel approach based on a combination of stem cell delivery, heat-inducible gene expression and mild heating with high-intensity focused ultrasound (HIFU) under MRI guidance to remotely permeabilize BBB. The permeabilization of the BBB will be controlled with, and limited to where selected pro-inflammatory factors will be secreted secondary to HIFU activation, which is in the vicinity of the engineered stem cells and consequently both the primary and secondary disease foci. This therapeutic platform thus represents a non-invasive way for BBB opening with unprecedented spatiotemporal precision, and if properly and specifically modified, can be clinically translated to facilitate delivery of different diagnostic and therapeutic agents which can have great impact in treatment of various disease processes in the central nervous system.

A primer on molecular biology for imagers: III. Proteins: structure and function.

This article along with the first 2 in this series (4,12) completes the discussion on the key molecules and process inside the cell namely, DNA, RNA, and proteins. These 3 articles provide a very basic foundation for understanding molecular biology concepts and summarize some of the work of numerous scientists over the past century. We understand these processes far better now than we did in the past, but clearly this knowledge is by no means complete and a number of basic scientists are working hard to elucidate and understand the fundamental mechanisms that operate within a cell. Genes and gene products work with each other in complex, interconnected pathways, and in perfect harmony to make a functional cell, tissue, and an organism as a whole. There is a lot of cross-talk that happens between different proteins that interact with various other proteins, DNA, and RNA to establish pathways, networks, and molecular systems as a team working to perfection. The past 15 years have seen the rapid development of systems biology approaches. We live in an era that emphasizes multi-disciplinary, cross-functional teams to perform science rather than individual researchers working on the bench on a very specific problem. Global approaches have become more common and the amount of data generated must be managed by trained bioinformatics personnel and large computers. In our subsequent articles, we will discuss these global approaches and the areas of genomics, functional genomics, and proteomics that have revolutionized the way we perform science.

A primer on molecular biology for imagers: II. Transcription and gene expression.

The process of gene expression is complex and highly regulated to ensure that the right gene is expressed at the right place, at the right time, and in regulated amounts. The cell has multiple levels at which it controls the expression of a transcript including gene expression, alternate splicing, and stability of the transcript. Alternate splicing to generate different RNA species from a given gene and DNA rearrangements where genes are rearranged during cellular differentiation (eg, immunoglobulin genes) are additional mechanisms used to generate diversity in complex organisms. Epigenetic mechanisms such as methylation where CpG-rich islands in the promoter region depending on their methylation status can also modulate gene expression. The reader is requested to refer to the books, review articles, and web sites for additional information.

Molecular imaging and therapy directed at the neovasculature in pathologies. How imaging can be incorporated into vascular-targeted delivery systems to generate active therapeutic agents.

We have discussed the impact of molecular imaging on clinical and preclinical medicine. We have presented the potential problems of delivering the effective therapeutic dose and the properties that can help contribute to the drug efficacy. The rationale for the design of new antiangiogenic agents that can be used for imaging and therapy was presented. Finally, results from imaging and targeted nanoparticle based therapies were presented. In vivo imaging of angiogenic tumors using anti-alpha(v)beta3 -targeted polymerized vesicles composed of the murine antibody LM609 attached to NPs labeled with the MR contrast agent gadolinium in the V2 carcinoma model in rabbits. MRI studies using this targeted contrast agent revealed large areas of alpha(v)beta3 integrin expression in tumor-associated vasculature that conventional MRIs failed to show. Other investigators have used microemulsions conjugated to an antibody targeted against alpha(v)beta as imaging agents. These materials also show contrast enhancement of tumor vasculature undergoing angiogenesis. Other markers, such as the PECAM-1 (CD-31), VCAM-1 (CD54) and VEGF receptor (flk-1), have been shown to be upregulated on tumor endothelium and associated with angiogenesis but have not been used in imaging studies. Furthermore, by modification of the NPs, we were able to use this imaging agent as an antiangiogenic gene delivery system. The results from these studies are very promising and are being further pursued.

A system mathematical model of a cell-cell communication network in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating and chronic neurodegenerative disease without any known cure. In the brain and spinal cord of both patients and animal models with ALS, neuroinflammation is a prominent pathological hallmark which is characterized by infiltrating T cells at sites of motor neuron injury. Their presence in mutant Cu(2+)/Zn(2+) superoxide dismutase (mSOD1) induced ALS plays an important role in shifting the response of microglia from neuroprotective to neurotoxic. In order to better understand how these cells and their communication network collectively modulate the disease progression, we have established a mathematical model integrating diverse cells and cytokines. According to the experimental data sets, we first refined this model by identifying a link between TGFß and M1 microglia which can produce an optimized model to fit data sets better. Then based on this model, parameters were estimated using genetic algorithm. Sensitivity analysis of these parameters identified several factors such as the release rate of IFNγ by T helper 1 (Th1) cells, which may be related to the heterogeneity between the patients with different survival times. Furthermore, the tests on T cell based therapeutic strategies indicated that elimination of Th1 cells is the most effective approach extending survival time. This confirmed the dominant role of Th1 cells in leading the rapid disorder in the later stage of ALS. For the therapies targeting cytokines, injection of IL6 can essentially augment the neuroprotective response and extend the life effectively by elevating the level of IL4, a neuroprotective cytokine, while directly injected IL4 will decay rapidly in the ALS microenvironment and cannot provide a persistent protective effect. On the other hand, in spite of the attractive effect of direct elimination of mSOD1 or self-antigen, it is difficult to implement in CNS. As an alternative, elimination of IFNγ can be chosen as another effective therapy. In the future, if we combine the side effects of different therapies, this model can be used to optimize the therapeutic strategies so that they can effectively improve survival rates and quality of life for patients with ALS.