Abstract

Lymphoseek, a receptor-targeted sentinel node mapping agent: a review and future investigations

Wallace AM, Me´ndez J, Ellner SJ, Hoh CK, Orahood RC and Vera DR

Lymphoseek, is a technetium-99 m-labeled radiotracer, that binds to a lymph node-specific receptor. Its small molecular diameter of 0.007 um provides significantly faster injection site clearance than filtered Tc- 99 m-sulfur colloid (fTcSC); its receptor binding property provides a molecular mechanism for high lymph node retention. FDA-approved Phase I clinical trials in melanoma and breast cancer were conducted to compare the injection site clearance, sentinel node accumulation, and safety profiles of Lymphoseek and fTcSC. Preclinical studies in pigs were conducted to measure colon and gastric sentinel lymph node (SLN) uptake. The injection site clearance half-life for a subcutaneous injection of Lymphoseek was 7-fold faster than the fTcSC clearance. The half-life of a peritumoral/ subdermal breast injection was 18-fold faster than a fTcSC injection. SLN uptake was equivalent for both radiotracers. Single dose intradermal Lymphoseek breast injections cleared the injection site twice as fast the periturmoral/subdermal injections. After an intradermal injection SLN accumulation was approximately 3-fold higher at 16 hours (next day protocol) than 3 hours (same day protocol). GI studies in pigs indicate that Lymphoseek provides a 5 to 180 min window of high SLN uptake with no distal node accumulation. Lymphoseek clearance half-lifes of colon and gastric injection sites were 4 times faster than fTcSC. Future investigations will include prostate cancer where Lymphoseek's rapid injection site clearance and sustained SLN uptake will permit intra-operative administration. We will also employ the receptor-binding technology to target specific cellular components within vaccinedraining sentinel lymph nodes; our goal will be the design of probes for directing adaptive immunotherapy.

How to prepare for progress over the next twenty-five years. A proposal

Hiroshi Tazaki

Over the last 10 years, the most prominent developments in clinical medicine were the introduction of gene therapy and minimally invasive surgery. Basic scientific progress was so rapid that practical activities, teaching young fellows, clinical training of residents were left behind. Thus, ethical problems in conducting new research projects and patient care, resulted in many legal and malpractice cases. In the future, medical and surgical technology will be progressing faster than in the last decade. Medical students and post-graduate residents must learn more, while the number of teachers with knowledge and technique declines. In addition, a complete human being with a wide general sense of the world outside of medicine is needed for medical science and patient care in the future. Medical students should attend a four-year college and go on to medical school after graduation-standard in medical schools in the United States for over a half-century. Nevertheless the above-mentioned problems exist in both post-graduate research studies and clinical training. If a medical school in Japan introduces this, Japanese medical science will attain world leadership over the next 25 years by combining the best ideas of the United States, while maintaining a Japanese standard of quality and technology.

Intestine-specific transcription factors regulate proliferation and cell-cell adhesion by modulation of b-catenin function

Toshihiko Ezaki, Rong-Jun Guo, Eun-Ran Suh and John P. Lynch

A critical event in human colon carcinogenesis is the disruption of b-catenin regulation, both free in the cytoplasm and cadherin-bound. The mechanisms normally used to regulate b-catenin function are not fully understood. We previously reported that Cdx2, an intestine- specific transcription factor, diminished colon cancer cell proliferation by blocking b-catenin/TCF transcriptional activity. We also reported that Cdx2 expression activates E-cadherin-mediated cell adhesion. We explored several potential mechanisms to explain these coordinated effects. We found that b-catenin, but not TCF4, can immunoprecipitate with Cdx2. The converse is true as well: Cdx2 immunoprecipitates with bcatenin but not TCF4. We also observed that this binding by Cdx2 disrupts the b-catenin/TCF interactions in a dose-dependent manner. Furthermore, using Cdx2 mutants, we determined that a critical domain required for b-catenin binding and disruption of b-catenin/TCF transcriptional activity resides in the N-terminus but outside of the transcription-activating (TA) domain. Studies of the induction of cell-cell adhesion by Cdx2 found that neither E-cadherin, a-, b-, nor g-catenin protein levels were significantly altered by Cdx2 expression. p120-catenin levels were modestly induced and relatively hypophosphorylated, however. As tyrosine phosphorylation of b- and p120-catenin is known to disrupt their binding with E-cadherin and weaken cellcell adhesion, we tested for this. We found that Cdx2 expression significantly reduced tyrosine phosphorylation on b- and p120-catenin proteins. An shRNA knockdown of PTP-1B, a candidate tyrosine phosphatase involved in this process, disrupted Cdx2 induced cell-cell adhesion and restored b- and p120-catenin tyrosine phosphorylation. We conclude that Cdx2 expression regulates colonocyte proliferation and cell-cell adhesion by altering b-catenin's role in these processes.

Allogeneic stem cell transplantation for Chronic Myelogenous Leukemia (CML) in the imatinib era

Richard T. Maziarz

Hematopoietic stem cell transplantation has long served as the primary therapy for age appropriate patients with chronic myelogenous leukemia (CML) and remains the only identified proven curative therapy, although late recurrences have been identified. Imatinib mesylate, a small molecule inhibitor of the deregulated BCR-abl tyrosine kinase, the molecular epicenter in the pathogenesis of CML, has revolutionized non-transplant therapy with CML and has displaced transplantation as the primary treatment for young patients. With the rise in imatinib utilization, there has been a reciprocal fall in allogeneic transplantation procedures for CML in the United States and worldwide, as recorded by the CIBMTR and other transplant registries. It still needs to be determined in the short and long term that transplantation outcomes will not have been compromised by the introduction of this new class of targeted therapies. In addition, the availability of reduced intensity allogeneic stem cell transplantation with its potential applicability to nearly all CML patients mandates critical review of all data surrounding the long-term efficacy of transplantation with constant vigilance in monitoring the potential for long-term success with imatinib. Numerous questions remain to be answered only through evolving clinical trials, yet decisions for individual patients need to be made based upon the best available data. Ongoing critical examination of optimal imatinib dosing and the clear definition of inadequate response and/or resistance as well as the potential application of the second generation BCR/abl kinase inhibitors, will allow one to best develop the algorithms for the determination for whom transplant should remain primary therapy or will allow the identification of those patients for whom transplantation should best be reserved for a future application.

Immunological and non-immunological risk factors in renal transplantation

Susan V Fuggle and Isabel Quiroga

There are many immunological and non-immunological factors that influence the outcome of renal transplantation, but the effects of factors relating to the donor have not been fully explored. The impact of a large number of donor and pre-transplant factors on transplant outcome was studied in cadaveric transplants performed at the Oxford Transplant Centre. The results of the analysis of well-matched transplant recipients showed that cold ischaemia time (CIT) and delayed graft function (DGF) are the most important predictors of poor short and long-term graft survival. In order to improve long-term transplant survival efforts should focus on limiting CIT and the damage that occurs during this period. Genetic factors influence transplant outcome and HLA matching has a beneficial effect, even with modern immunosuppression. There are other polymorphic genes with immunological function including the stress induced MHC class I-related chain genes (MICA, MICB). Immunohistochemical studies were performed to determine the expression of MICB in renal transplant biopsies. Variable tubular MICB expression was evident in donor biopsies and following transplantation induction of MICB was associated with histological evidence of renal cellular stress. Donor specific anti-MICA antibodies have been detected following rejection of renal transplants. Deposition of the complement protein C4d in renal allograft biopsies obtained during graft dysfunction has been proposed as a marker of antibody-mediated rejection, but it is important to determine the diagnostic specificity of C4d deposition. Our results from protocol biopsies show that C4d is specific for humoral rejection, as indicated by its association with donor-reactive antibodies and acute rejection.

Imaging of brain microcirculation in ischemia and transplanted adult stem cells

Jacques Seylaz

To explore the cortical microcirculation of rats and mice in vivo and in real-time, we developed a new method using laser-scanning confocal fluorescence microscopy through a closed cranial window. The videomonitoring of fluorescently labelled red blood cells enables the investigation of changes in capillary circulation at 200-mm-depth in the cortex of animals placed under the confocal microscope. This method was used in rats to explore red blood cells flow through capillaries during post-ischemic cortical hyperemic reactions to transient global ischemia, and to relate them to microvessel diameter changes. The method was then adapted to mice, and a cranial window was elaborated for chronic use in repeated imaging sequences over several weeks. To study microcirculatory events within the core of ischemia, a new strategy was designed to induce ischemia by thermocoagulating distal branches of the middle cerebral artery directly through the cranial window. Finally, as a first step in experimental cell therapy in the brain, we used this method to investigate in vivo the fate of various adult stem cells transplanted into the mouse ischemic brain. Green fluorescent protein (GFP)-expressing cells were stereotaxically injected into the cortex of mice with focal cerebral ischemia. The data demonstrated that adult stem cells of various origins survived and migrated to the ischemic and periischemic areas, often with a peri-vascular location, and modified their phenotype. Such an approach indicates that adult stem cells could be used as vehicles to introduce therapeutic genes into the central nervous system in an attempt to support brain recovery.

Molecular pharmacology of T-type Ca²⁺ channels

Edward Perez-Reyes

Calcium enters the cytosol either through plasma membrane ion channels, or is released from intracellular pools. Rises in intracellular calcium then trigger a wide range of physiological processes ranging from muscle contraction to neurotransmitter release. Plasma membrane ion channels are typically opened after depolarization of the plasma membrane, and hence are called voltage-gated channels. The diversity of voltagegated calcium channels has been extensively studied using electrophysiological, biochemical, pharmacological, and more recently using molecular biology techniques. The main channel protein is called the alpha-1 subunit, which are encoded by 10 genes. Alignments reveal that there are three main subfamilies of a1 subunit: Ca_v1, which contains four members that encode L-type channels; Ca_v2, which contains three members including the P/Q-, N-, and R-types; and Ca_v3, which contains three members that encode T-type channels. Drugs that block L-type channels are useful in the treatment of high blood pressure, angina, and arrhythmia. A hot area of research is the development of novel calcium channel blockers. The pharmacology of T-type channels will be reviewed, and predictions will be made on the utility of a highly selective blocker Tchannel blocker.