Abstract

Last Updated on Monday, 29 June 2009 09:00

THERAPY OF PULMONARY ASPERGILLOSIS IN INFECTED MICE WITH ANTIBODY-ALLIINASE CONJUGATES AND ALLIIN.

David Mirelman, PhD, Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.     This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Aspergillus species are ubiquitous soil microorganisms whose conidia we inhale on a regular basis and they are usually harmless to immuno-competent individuals. In immuno-compromised patients the mortality caused by pulmonary Aspergillosis has been shown to reach >50 %. Numerous strategies to treat these infections have been reported, most of them are based on different formulations and applications of Amphotericin B, and are mainly designed to overcome the high toxicity of this antifungal agent.  We have produced an anti-Aspergillus fumigatus (AF) monoclonal antibody, which binds to AF at nanomolar concentrations.  Conjugates of this m-antibody were prepared by chemical ligation with the Garlic enzyme Alliinase (1) which efficiently catalyzes the synthesis of Allicin (diallyl-thio-sulfinate) from the natural substrate Alliin, (S-Allyl-cysteine-sulfoxide). Allicin is a hydrophobic molecule best known as Nature’s antibiotic which rapidly penetrates through biological membranes and possesses a wide range of antimicrobial activities (2).  The mode of action of Allicin is through the modification of free thiols (conversion of -SH groups into –S-S-) resulting in the inhibition of many thiol containing enzymes (3-5). In the presence of Alliin, the anti Aspergillus mAb-Alliinase conjugates generated a fungicidal activity which killed a variety of Aspergillus  isolates at a concentration as low as 10 nmol. In vivo, the mAb-Alliinase conjugates in the presence of Alliin, specifically killed the fungi and completely cleared lungs of immuno-suppressed infected mice from Aspergillus infection. Intratracheal treatment with mAb-Alliinase/Alliin resulted in 80%-90% survival of mice with total recovery and complete fungal clearance. Intratracheal repetitive treatments with the mAb-conjugate + Alliin were effective not only when started on the day of infection, but also when applied at a more a advanced stage (50 h) after infection and spread of the fungal disease.  No toxic effects were noticed although in some case an aspergilloma was detected. Alliinase alone or non conjugated antibodies, without Alliinase, failed to protect mice from aspergillosis. These findings indicate that Allicin has a remarkable fungicidal activity in vivo and is very effective when produced on the surface of conidia whereas when it is produced without targeting to the Aspergillus cell surface, Allicin, which is a very reactive and short lived molecule, was not effective.


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1. Arditti, FD, Rabinkov, A. Mirelman, D. et al.   Mol. Cancer Therapy  4: 325-331, 2005
2 . Schadkchan Y., Shemesh, E. Mirelman, D. et al.   Antimicrobial Chemotherapy, 53, 832-836, 2004
3 T.  Miron, A. Rabinkov, D. Mirelman, et.al  , Biochem. Biophys. Acta,  1463, 20-30, 2000
4.  Miron T, Sivaraman H, Rabinkov A, Mirelman D, Wilchek M. Anal Biochem. 351(1):152-154, 2006
5 Prager-K   M. , Rabinkov A. , Mirelman , D et al .  Cell Motility and Cytoskeleton, 64, 321-337,2007

 

STRUCTURAL STUDIES OF GENE TRANSFER BY AGROBACTERIUM TUMEFACIENS

Micheal Elbaum, Ph.D.
Department of Materials and Interfaces, The Weizmann Institute of Science, Rehovot 76100, Israel

Agrobacterium tumefaciens causes “crowngall” disease in many flowering plants. It infects its hosts by a mechanism involving gene transfer and ultimately genetic modification. It has been widely adopted by plant scientists as a vehicle for artificial transformation. The process is similar to bacterial conjugation, yet the transferred DNA moves between very different cytoplasmic environments from prokaryote to eukaryote.
This inter-kingdom transfer requires an adapter, in the form of A DNA-binding protein known as VirE2. We have studied VirE2 by Electron microscopy, with image processing and three-dimensional reconstruction, and by X-ray crystallography. These reveal a multi-domain protein with a novel fold. The overall architecture resembles several disease effectors of other species. This suggests that the unique capacity of Agrobacterium for inter-kingdom gene transfer can be explained as a convergence of conjugation and toxin secretion, where the toxin is not toxic per se but acts to protect the DNA en route to the host nucleus.
 
PHARMACOLOGICAL STIMULATION OF FETAL HEMOGLOBIN AS A THERAPEUTIC MODALITY FOR HEMOGLOBINOPATHIES  
E. Fibach, Dept. of Hematology, Hadassah – Hebrew University Medical Center, Jerusalem, Israel.
Tel: 972-2-6776751; Fax: 972-2-6423067; e-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

Mutations in the -globin gene, which result in -hemoglobinopathies (sickle cell anemia and -thalassemia), are globally the most common class of inherited single gene disorders. Current treatment options are limited, especially in developing countries; more practical and cheaper therapies are urgently needed. Since it has been shown that high fetal hemoglobin (HbF) ameliorates the clinical symptoms of these diseases, one of the currently most studied approaches involves attempts to reactivate the -globin genes and stimulate the production of HbF by pharmacological agents. Hydroxyurea is currently the only HbF-promoting drug in clinical use. It is, however, a cytotoxic drug with serious side-effects, including carcinogenic and teratogenic potentials. Therefore, the search for more potent and less toxic drugs continues. Several in vitro and in vivo experimental models have been developed for this purpose. The models include in vitro established erythroid-like cell lines and primary cultures (both in semi-solid and liquid media) of erythroid cells derived from progenitors obtained from normal donors and the patients; as well as in vivo models in genetically modified (transgenic) and unmodified animals. These experimental models are useful for large-scale screening of compounds for HbF-stimulating potential, for studying their mechanism of action at the cellular and molecular levels, and for studying the pharmacology, pharmacokinetics and toxicology of the drugs. These studies are essential for finding, testing and developing of new drugs that will be effective and safe for clinical use in adults and children.


 

MODERN AGRICULTURE IN ISRAEL – RESEARCH,  DEVELOPMENT AND TECHNOLOGY TRANSFER

Sara Spiegel, Agricultural Research Organization (ARO) - The Volcani Center, Bet Dagan 50250, Israel   Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

On-going research, development (R&D) and technology transfer are major factors which contributed to the advancement of the agricultural industry in Israel. Among the institutes, universities and centers involved in agricultural research in Israel, Agricultural Research Organization (ARO), the research arm of the Ministry of Agriculture and Rural Development is responsible for planning, conducting and implementing the majority of the agricultural R&D effort in the country. ARO has an extensive research infrastructure that supports both applied and basic research conducted by ~200 PhD scientists and 300 engineers and technicians organized in research teams. Research is conducted in six institutes located on the main campus at Bet Dagan and in two regional research centers in the north (Newe Ya'ar) and south (Gilat) of the country.
The major research areas covered by ARO include various aspects of plant and animal sciences, soil, water and environment, plant protection, post harvest technology and engineering. Research at ARO is aimed at improving environmentally responsible agricultural production systems, introducing new products, processes and labor-saving equipment.
 A multidisciplinary approach, bringing together scientists from various areas resulted in significant results for the benefit of farmers and consumers. Issues like desertification, water scarcity, post harvest storage of fresh produce have been targeted at ARO. Research projects are either on a country level, bilateral agreements, or in frames addressing international collaboration with other countries, both developed and developing.
Dissemination of research results to end-users has been conducted in various ways for the benefit of farmers and consumers. Developed technologies with a commercialization potential are handled by an autonomous unit ('Kidum') within the ARO. This unit identifies projects with a business potential; negotiates R&D and licensing agreements and handles the Intellectual Property of ARO.  
Participation of individual graduate students or young scientists from various countries in research projects at ARO has been practiced and allows visiting scientists to expand their research experience.
 
 

DYNAMIC INTERACTIONS BETWEEN THE NERVOUS AND IMMUNE SYSTEMS WITH THE MICROENVIRONMENT, REGULATE NORMAL AND LEUKEMIC HEMATOPIETIC STEM CELLS

By Tsvee Lapidot Ph.D, Dept. of Immunology Weizmann Institute. Rehovot, Israel.

The hallmark of hematopoietic stem cells is their motility, self-renewal and developmental potential. Blood forming stem cells are defined in transplantation assays based on their functional ability to migrate to the recipient bone marrow and to durably repopulate it with immature and maturing leukocytes that continuously replenish the blood circulation. Functional, preclinical models for normal and leukemic human stem cells were developed using immune deficient NOD/SCID mice as recipients. These models identified normal human SCID repopulating stem cells as well as SCID leukemia initiating stem cells, obtained from some myeloid (AML) and lymphoid (Pre B ALL) leukemic ptients. Engraftment kinetics and repopulation potential of human AML and Pre B ALL stem and progenitor cells in transplanted immune deficient mice can predict clinical outcome. The chemokine SDF-1 is the only powerful chemotactic factor for both human and murine stem cells, which functionally express its major receptor CXCR4. Engraftment and repopulation of immune deficient mice by normal and leukemic human stem cells are dependent on SDF-1/CXCR4 interactions, since human and murine SDF-1 are cross reactive and differ in only one amino acid. Homing, retention, release and stem cell mobilization are tightly regulated processes, which involve bone turnover and an interplay between cytokines, chemokines, adhesion molecules and proteolytic enzymes. The roles of CD44, MT1-MMP and RECK in stem cell migration will be discussed.

Most blood forming stem cells are retained in the bone marrow (BM), anchored to specialized niches via adhesion interactions, which prevent their motility and proliferation. However, low levels of motile progenitors migrate in the circulation as part of homeostasis. These low levels are dramatically amplified during alarm situations in response to stress signals due to injury, bleeding and infections, as part of host defense and repair mechanisms. These stress signals are mimicked  by repeated G-CSF stimulation in order to mobilize stem and progenitor cells to the circulation in order to harvest them for clinical transplantation protocols.

Stem cell adhesion interactions with the stromal niche supporting cells need to be dynamic in order to allow the undifferentiated cells to proliferate, differentiate and migrate. Osteoclasts have a dual role in host defense: bone remodeling and regulation of stem cells by freeing them form their inhibitory anchorage to stromal cells. Thus, osteoclast/osteoblast interactions also regulate BM leukocyte production on demand. Both osteoclasts, osteoblasts and stem and progenitor cells functionally express receptors for neurotransmitters. Immature human CD34+ cells dynamically express dopamine and epinephrine receptors and inflammatory, myeloid cytokines such as G-CSF and GM-CSF increase catecholaminergic receptor expression in order to facilitate leukocyte production and trafficking. This up regulation activates the progenitor cells in response to stimulation by the neurotransmitters and induces their motility and proliferation via Wnt signaling. In conclusion, regulation of leukocyte production and trafficking by stem cells in the BM reservoir is dynamic and involves mutual, reciprocal interactions between the nervous and immune systems with the stromal microenvironment throughout the body.

Both normal and leukemic human stem and progenitor cells functionally express neurotransmitter receptors, which are involved in regulation of their motility and proliferation. In summary, stem cells are directly and indirectly regulated by dynamic interactions of the nervous and immune systems with the microenvironment.

 

MECHANISMS UNDERLYING THE REGULATION OF ANTIGENIC VARIATION IN THE MALARIA PARASITES PLASMODIUM FALCIPARUM.

Ron Dzikowski
Department of Microbiology & Molecular Genetics, Institution for Medical Research Israel-Canada (IMRIC), Hebrew University Hadassah Medical School.


Pathogens of the genus Plasmodium are unicellular parasites that infect a variety of animals, including reptiles, birds, and mammals. All Plasmodium species target host erythrocytes and replicate asexually within this niche.  In humans, proliferation within erythrocytes causes disease symptoms ranging from asymtomatic infection to severe disease, including mild to severe febrile and respiratory symptoms, profound anemia and obstruction of blood flow. The most serious form of human malaria is caused by P. falciparum, a pathogen that is responsible for several million deaths annually throughout the developing world. Malaria parasites succeed in evading the host immune response to establish long term, persistent infections, thus increasing the efficiency by which they are transmitted to the mosquito vector. The ability to evade the host immune system, in particular the avoidance of antibody-mediated immunity against parasite-encoded surface proteins, is the result of amplification of extensive repertoires of multi-copy, hypervariable gene families that encode infected erythrocyte or merozoite surface proteins. Via switching between antigenically diverse genes within these large families, populations of parasites have the capacity for rapid variation in antigenicity and virulence over the course of an infection. We will discuss the mechanisms underlying their tightly controlled gene expression and antigenic switching.

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