Dorel L. Radu

CD44s-targeted treatment with monoclonal antibody blocks intracerebral invasion and growth of 9L gliosarcoma.

Glioma invasiveness is a complex process involving recognition and attachment of tumor cells to particular extracellular matrix (ECM) molecules prior to migrating into proteolytically modified matrix and inducing angiogenesis. CD44 is a group of transmembrane adhesion molecules found on a wide variety of cells including gliomas that has been suggested as the principal mediator of migration/invasion. The aim of the present study was to demonstrate whether antibody specific for the standard form of CD44 (CD44s, 85–90xa0kDa) might prevent invasion, thus blocking growth of the 9L gliosarcoma inxa0vivo. High expression of CD44s on the surface of 9L cells and brain tumors was demonstrated by immunochemistry. Fluorescence-activated cell sorting (FACS) demonstrated binding saturation of anti-CD44s monoclonal antibody (mAb) to the receptor at 1xa0μg/5 × 105 cells. Blocking of CD44s in vitro resulted in a dose-dependent progressive, up to 95% ± 2.5% detachment of 9L cells from ECM-coated culture surfaces. Blocking of CD44s in vivo resulted in significantly reduced 9L brain tumors (2.5% ± 0.4%) – measured as the quotient: tumor surface (mm2)/brain surface (mm2) × 100 – as compared to untreated (16.1% ± 2.2%) or sham-treated rats (16% ± 3.7% to 16.1% ± 3%). We conclude that CD44s-targeted treatment with specific mAb may be an effective means for preventing glioma progression.

Escape from Self-tolerance Leads to Neonatal Insulin-dependent Diabetes Mellitus

Double transgenic (dTg) mice expressing the hemagglutinin (HA) of influenza virus under the insulin promoter and the TCR specific for the immunodominant CD4 T cell epitope of HA (HA110-120) develop insulin-dependent diabetes mellitus (IDDM). In order to gain information on the breaking down of neonatal self-tolerance we studied the occurrence of IDDM after birth. Our results showed that newborn mice develop fulminant IDDM characterized by occurrence of insulitis as early as 3 days after birth, followed by hyperglycemia by 7 days, and significant hypoinsulinemia by 28 days. The neonatal breakdown of self-tolerance of T cells positively selected in the thymus is supported by the facts that: (i) peripheral HA110-120 specific T cells from neonates are fully functional and proliferated upon stimulation with the nominal peptide, and (ii) peptide-specific T cells were accumulated in the pancreas of dTg mice as early as 3 days after birth. Our results demonstrate that diabetes occurring in young dTg mice is due to early activation of self-reactive T cells immediately after birth. Accumulation of specific T cells in the target organ leads to destruction of pancreatic beta-cells and IDDM. These mice may provide a useful model to evaluate new strategies for the prevention of diabetes.

Expression of activation‐induced cytidine deaminase decreases throughout the life

Activation‐induced cytidine deaminase (AID) is an RNA editing enzyme, which contributes to generation of new functional genes from a restricted number of genes of plant and animal genome. This enzyme was involved in the process of somatic mutation and class switching in vertebrate. Since the rate of somatic mutations is variable throughout ontogeny, we have studied the transcription of AID in 3 to 24 month‐old Balb/c mice. Our results demonstrate a significant decrease of the transcription of the AID gene with aging. The decreased AID activity is not related to variation of phenotypic and functional properties of B cells throughout the life. This observation can explain the low rate of somatic mutation in aged animals.

Maintenance of size and function of influenza virus hemagglutinin specific transgenic T‐cell clone during life

Immunization induces less protective immunity against infectious diseases in old compared to young subjects. We have studied the effect of age on the in vitro and in vivo function of murine transgenic T cells expressing a receptor for influenza hemagglutinin 110‐120 peptide. During aging the transgenic T cells undergo the age‐associated shift from naive to memory phenotype but maintain, despite thymic involution, their number as well as their cytokine production and proliferative responses induced by the hemagglutinin 110‐120 peptide in vitro. The maintenance of the size and functions of transgenic T cells during the aging may be related to low expression of CTLA‐4 molecules known to exhibit a negative regulatory effect subsequent to interaction with costimulatory molecules as well as of stimulation of T cells by unknown cross reactive endogenous factors but not by nominal antigen since innate immunity prevents natural infection with influenza virus of murine species. This suggests that the impaired immunity induced by immunization in old subjects reflects defects in the development and maintenance of T cell memory and not in the expression of effector activity.

Cellular and molecular studies of B cells exhibiting reverse somatic mutation throughout life

Somatic mutation of immunoglobulin (Ig) genes plays an important role in generating antibody diversity. The frequency of somatic mutation appears to vary throughout life. However, this process has been difficult to study in vivo because the DNA in and around rearranged V genes undergoes random mutation, causing silent or replacement mutations. Therefore, we have developed a transgenic mouse model for studying the frequency of B cells exhibiting mutation in young and old mice. The system is based on a reporter transgene (HuG‐X) that encodes a chimeric Ig heavy chain composed of a murine VDJ segment and a human IgG1 constant region. The VDJ has been mutated to contain a TAG stop codon in the D segment. Therefore, the transgene is transcribed but not translated. Point mutation of the stop codon results in expression of the chimeric H chain, which is readily detected as human IgG1 expression. In vivo, we found that the transgene undergoes spontaneous reverse somatic mutation at a low frequency. Treatment of HuG‐X mice with anti‐IgD greatly increases the frequency of somatic mutation. The observed mutation frequency in anti‐IgD‐treated mice increases with age until adulthood, then plateaux and finally declines in aged mice. The mutations in the stop codon were associated with increased double‐stranded DNA breaks (DSB) within and around the TAG site. Our results demonstrate that the rate of frequency of spontaneous reverse mutation is very low in vivo, yet it is significantly increased after stimulation with anti‐IgD antibodies. The frequency of point mutation is age dependent and correlates with increased DSB.