Potent inhibitors of the MAP kinase p38

The MAP kinase p38 plays a key role in the biosynthesis of the inflammatory cytokines TNF-alpha and IL-1. We have developed a novel series of potent p38 inhibitors that could lead to new methods of treatment for inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease.     

Reduction in new metastases in breast cancer with adjuvant clodronate treatment

We report an unusual case of peripheral primitive neuroectodermal tumor (pPNET) in an infant presenting with congenital cutaneous nodules and a t(7;22)(p21;q11.2). The biologic behavior of the tumor diverged over time from a slowly growing tumor with multiple cutaneous nodules to a more aggressive neoplasm characterized by pulmonary metastases and a soft tissue mass showing additional cytogenetic alterations.     

CRP-mediated activation of complement in vivo: assessment by measuring circulating complement-C-reactive protein complexes

The in vivo function of C-reactive protein (CRP) is unknown. Among the in vitro functions assigned to CRP is the ability to activate complement via the classical pathway. To date, there is no evidence supporting that CRP exerts this function in vivo. We here show a novel approach to assess CRP-mediated complement activation in vivo, which is based on the property that activated complement factors C3 and C4 fix to CRP during complement activation induced by this acute phase protein. We developed specific ELISAs for complexes between CRP and C4b, C4d, C3b, or C3d. We established that in vitro complement-CRP complexes were formed only during CRP-dependent activation, and not during activation by other activators, even in the presence of high CRP levels. Circulating levels of complement-CRP complexes were undetectable in normal donors, but significantly increased in nine patients following implantation of a renal allograft. Importantly, levels of complement-CRP complexes did not change in these patients upon a bolus infusion of mAb OKT3, which induces activation of the classical complement pathway, demonstrating in vivo that complement-CRP complexes are not formed during CRP-independent activation of complement, even when CRP is elevated. We conclude that measurement of complement-CRP complexes provides a suitable tool to study CRP-mediated activation of complement in vivo. Furthermore, increased levels of these complexes occur in clinical samples, indicating that CRP may induce activation of complement in vivo.     

NGF involvement in pain induced by chronic constriction injury of the rat sciatic nerve

Chronic constriction injury (CCI) of the rat sciatic nerve, which within 3 days induces thermal and mechanical hyperalgesia and mechanical allodynia, is used as a model for pain resulting from nerve injury. Involvement of nerve growth factor (NGF) in the development of this hyperalgesia is suggested by the increase in the level of mRNA encoding NGF in cells in the injured area and in dorsal root ganglia at the level of the lesion and the greatly increased NGF levels (determined by ELISA) in the ganglia ipsilateral to the CCI. Application of anti-serum to NGF at the site of CCI delayed the appearance of hyperalgesia, whereas pre-immune serum appeared to enhance it. These results are consistent with the view that NGF is an important factor in the appearance of hyperalgesia associated with unilateral mononeuropathy.     

Adenovirus vector-based purging of multiple myeloma cells

Adenoviruses are efficient gene delivery agents for a variety of neoplasms. In the present study, we have investigated the use of adenoviruses for the delivery of the thymidine kinase (tk) gene into multiple myeloma (MM) cells. We first demonstrated that MM cell lines and MM patient cells express both adenovirus receptors as well as the DF3/MUC1 protein, thus providing a rationale for using adenoviruses to selectively deliver genes under the control of the DF3 promoter. By using an adenoviral construct containing beta-galactosidase (beta-gal) gene driven by the DF3 promoter (Ad. DF3-betagal), we demonstrate greater than 80% transduction efficiency in OCI-My5 and RPMI 8226 MM cell lines at a multiplicity of infection of 1 to 100. Importantly, transduction with the tk gene driven by the DF3 promoter (Ad.DF3-tk) followed by treatment with 50 micromol/L ganciclovir (GCV) purged >/=6 log of contaminating OCI-My5 and RPMI 8226 MM cells within bone marrow mononuclear cells. In contrast, normal human hematopoietic progenitor cell number was unaffected under these conditions. Selectivity of DF3/MUC1 promoter was further confirmed, because Ad.DF3-betagal or Ad.DF3-tk did not transduce MUC1-negative HeLa cervical carcinoma cells. In addition, GCV treatment of Ad.DF3-tk-transduced RPMI 8226 MM cells did not induce a significant bystander effect. These findings demonstrate that transduction with Ad vectors using a tumor-selective promoter provides a highly efficient and selective approach for the ex vivo purging of MM cells.     

Suppression of tumorigenicity and metastasis of human renal carcinoma cells by infection with retroviral vectors harboring the murine inducible nitric oxide synthase gene

The purpose of this study was to determine whether retrovirus-mediated transfer of the murine macrophage inducible nitric oxide synthase (iNOS) gene can inhibit tumorigenicity and metastasis of human renal cancer cells. Retroviral vectors encoding murine macrophage iNOS were constructed in the pLXSN retroviral vector with the iNOS gene under the control of a long terminal repeat promoter and a neomycin resistance gene under the control of an internal simian virus 40 promoter. Highly metastatic human renal carcinoma SN12PM6 cells were infected with control or iNOS retrovirus. Expression of iNOS was confirmed by Northern and Western blot analyses, and expression of the functional iNOS protein, i.e., production of nitric oxide (NO), was determined by measuring nitrite accumulation in culture supernatants. Noninfected or control cells produced large orthotopic tumors in the kidney of nude mice and a larger number of experimental lung metastases, whereas iNOS-infected cells produced small tumors in the kidneys and few to no lung metastases. The data indicate that the infection of human renal cancer cells by retroviruses harboring the murine iNOS gene can induce the production of high levels of NO, which is associated with autocytotoxicity, suppression of tumorigenicity, and abrogation of metastasis.     

Investigative techniques in the assessment of oral-pharyngeal dysphagia

Vascular disease is more commonly being studied at a molecular level. The knowledge derived is being applied to our understanding of clinical vascular disease processes and reconstruction outcome. Furthermore, applications are increasingly found for diagnosis and even intervention. The fundamental concepts and techniques involved in molecular biology are presented.