Lorri A. Morford

Immune defects observed in patients with primary malignant brain tumors.

Malignant glioblastomas (gliomas) account for approximately one third of all diagnosed brain tumors. Yet, a decade of research has made little progress in advancing the treatment of these tumors. In part this lack of progress is linked to the challenge of discovering how glial tumors are capable of both modulating host immune function and neutralizing immune-based therapies. Patients with gliomas exhibit a broad suppression of cell-mediated immunity. The impaired cell-mediated immunity observed in patients with gliomas appears to result from immunosuppressive factor(s) secreted by the tumor. This article reviews what has been elucidated about the immune defects of patients harboring glioma and the glioma-derived factors which mediate this immunosuppression. A model involving systemic cytokine dysregulation is presented to suggest how the immune defects arise in these individuals.

The oncofetal gene glypican 3 is regulated in the postnatal liver by zinc fingers and homeoboxes 2 and in the regenerating liver by alpha‐fetoprotein regulator 2

The Glypican 3 (Gpc3) gene is expressed abundantly in the fetal liver, is inactive in the normal adult liver, and is frequently reactivated in hepatocellular carcinoma (HCC). This reactivation in HCC has led to considerable interest in Gpc3 as a diagnostic tumor marker and its possible role in tumorigenesis. Despite this interest, the basis for Gpc3 regulation is poorly understood. On the basis of the similarities between Gpc3 and alpha‐fetoprotein expression in the liver, we reasoned that common factors might regulate these 2 genes. Here we identify zinc fingers and homeoboxes 2 (Zhx2) as a regulator of Gpc3. Mouse strain–specific differences in adult liver Gpc3 messenger RNA levels and transgenic mouse studies indicate that Zhx2 represses Gpc3 expression in the adult liver. We also demonstrate that Gpc3 is activated in the regenerating liver following a carbon tetrachloride treatment and that the level of Gpc3 induction is controlled by alpha‐fetoprotein regulator 2 (Afr2). Conclusion: We show that Zhx2 acts as a repressor of Gpc3 in the adult liver, and this raises the interesting possibility that Zhx2 might also be involved in Gpc3 reactivation in HCC. We also show that Gpc3 is activated in the regenerating liver in an Afr2‐dependent manner. Zhx2 and Afr2 represent the first known regulators of Gpc3. (HEPATOLOGY 2007.)

Differential localization of IL-2- and -15 receptor chains in membrane rafts of human T cells

We studied whether cytokine receptors (Rs) on T cells associate with lipid microdomains (“rafts”). Low‐dose phytohemagglutinin (PHA)‐stimulated human T cells were separated into cytoplasmic, membrane, and raft fractions by buoyant density centrifugation. Examination of these fractions for the presence of interleukin (IL)‐2‐ and ‐15R chains and associated signaling molecules by Western blotting revealed marked, selective enrichment of the IL‐2/15R β‐chain in rafts before IL‐2 stimulation. After IL‐2 stimulation, a substantial amount of the β‐chain was found in the membrane fraction. This partial translocation was also observed for the β‐chain‐associated molecules JAK‐1, p56lck, and grb‐2. Finally, raft disruption with methyl‐β‐cyclodextrin (MBCD) attenuated IL‐2‐induced tyrosine phosphorylation events and selectively decreased the surface expression of the IL‐2/15R β‐chain detected by flow cytometry. These results show that the IL‐2/15R β‐chain is enriched in rafts obtained from low‐dose, PHA‐stimulated T cells, that IL‐2 binding alters this enrichment, and that this enrichment may be functionally relevant as a possible mechanism to ensure cytokine selectivity and specificity.

Calpain II colocalizes with detergent-insoluble rafts on human and Jurkat T-cells.

Calpain, a calcium-dependent cysteine protease, is known to associate with the T-cell plasma membrane and subsequently cleave a number of cytoskeletal-associated proteins. In this study, we report the novel observation that calpain II, but not calpain I, associates with membrane lipid rafts on human peripheral blood T-cells and Jurkat cells. Raft-associated calpain activity is enhanced with exogenous calcium and inhibited with calpeptin, a specific inhibitor of calpain activity. In addition, we demonstrate that calpain cleaves the cytoskeletal-associated protein, talin, during the first 30-min after cell stimulation. We propose that lipid raft associated-calpain II could function in early TCR signaling to facilitate immune synapse formation through cytoskeletal remodeling mechanisms. Hence, we demonstrate that the positioning of calpain II within T-cell lipid rafts strategically places it in close proximity to known calpain substrates that are cleaved during Ag-specific T-cell signaling and immune synapse formation.

Insulin-like growth factors (IGF) enhance three-dimensional (3D) growth of human glioblastomas.

Human glioblastomas (gliomas) are characterized as rapidly growing brain tumors which are highly invasive but rarely metastatic. Human gliomas synthesize and secrete increased levels of insulin-like growth factors (IGFs) as well as expressing increased numbers of IGF receptors when compared to normal brain tissue. These observations suggest the existence of an IGF-mediated autocrine mechanism for glioma growth regulation. The purpose of this study was to examine the effect of human recombinant IGF (hrIGF) treatment on the in vitro growth of human glioma monolayer and three-dimensional (3D) multicellular spheroid cultures. The data demonstrate that hrIGF-I treatment of glioma cell lines slightly enhanced tumor monolayer proliferation as measured by [(3)H]thymidine incorporation. In contrast, treatment of glioma spheroids with hrIGF-I or hrDes(1-3)IGF-I, the truncated brain form of IGF-I, dramatically enhanced 3D tumor growth with a 1.5-2-fold reduction in spheroid doubling time (FRSDT). In addition, IGF-treated glioma spheroids were more densely packed than spheroids grown in media alone with no observed necrosis. These data suggest that IGFs will dramatically enhance glioma proliferation when 3D cell-cell contact occurs. This observed enhancement suggests that IGFs both synthesized in the brain and systemically support rapid proliferation of gliomas in vivo.

Functional characterization of the insulin-like growth factor I receptor on Jurkat T cells

Insulin-like growth factor I (IGF-I) has been shown to be important in the maintenance, development, and proliferation of various types of leukocytes, particularly T cells. Radio-receptor binding assays demonstrate that Jurkat T cells bind 125I-IGF-I with an affinity of 1.77 nM (Kd) and express approximately 230 receptors/cell. Specificity studies show insulin also binds the IGF-I receptor with an affinity 20-fold lower than that of IGF-I. Interaction of IGF-I with its receptor on Jurkat T cells induces the phosphorylation of tyrosine kinase which is detectable by Western blotting. The 95,000 MW protein detected is equivalent to the molecular weight of the beta chain of the IGF-I receptor described in other types of cells. These studies characterize the binding of IGF-I to its receptor on Jurkat T cells, demonstrate that IGF-I binding induces tyrosine phosphorylation, and support the hypothesis that IGF-I is important in the induction of T cell activation.

[8] Preparation, characterization, and use of human and rodent lymphocytes, monocytes, and neutrophils

Publisher Summary This chapter describes some simple, concise protocols for the preparation, characterization, and use of leukocytes. The model systems used to examine the role of the nervous and endocrine systems in degenerative and inflammatory disease processes have ranged from simple in vitro proliferative assays to complex in vivo systems. Much of the progress that has been made in understanding the mechanisms of neuroimmunomodulation has come from in vitro studies using primary cultures of leukocytes. The chapter also describes methods that can be used to prepare leukocytes for in vitro studies, as well as methods for lymphocyte activation and analysis of second-messenger production. Several factors should be considered when designing a set of experiments. One of the most important is context.

Bone Density and Dental External Apical Root Resorption

When orthodontic patients desire shorter treatment times with aesthetic results and long-term stability, it is important for the orthodontist to understand the potential limitations and problems that may arise during standard and/or technology-assisted accelerated treatment. Bone density plays an important role in facilitating orthodontic tooth movement (OTM), such that reductions in bone density can significantly increase movement velocity. Lifestyle, genetic background, environmental factors, and disease status all can influence a patients’ overall health and bone density. In some individuals, these factors may create specific conditions that influence systemic-wide bone metabolism. Both genetic variation and the onset of a bone-related disease can influence systemic bone density and local bone density, such as observed in the mandible and maxilla. These types of localized density changes can affect the rate of OTM and may also influence the risk of unwanted outcomes, i.e., the occurrence of dental external apical root resorption (EARR).

Genetic Factors Affecting Facial Growth

Malocclusion is the manifestation of complex genetic and environmental interactions on the development of the oral-facial region. Historically, orthodontists have been interested in genetics as a means to better understand why a patient has a particular occlusion, and to determine the best course of treatment for the malocclusion. The application of genetic information in treatment, however, has been hampered by several factors including: 1) the presumption that heritability studies have some clinical relevance to the individual patient, which they do not (Harris, 2008); 2) the presumption that whatever genetic factors may have contributed to the occlusion will also affect how the patient responds to treatment, which they may not; and 3) a lack of understanding to the extent at which genetic factors may interact with environmental factors (such as those created during orthodontic and dentofacial orthopedic treatments) to influence single gene (Mendelian) traits versus “Complex” traits which are more frequently observed in the clinic. (Hartsfield, 2011)

Genetic Implications in Orthodontic Tooth Movement

In orthodontics there is an interest in understanding how orthodontic tooth movement (OTM) may be modified with the use of differential anchorage and decreasing treatment time(s). While the focus of these efforts has been on how various procedures or devices affect OTM (i.e., typically increase OTM), there has been little discussion of how the patient’s genetic background may influence variation in OTM. In this chapter, the clinician will be introduced to basic concepts of clinical genetics to gain insight into various genetic factors that influence bone modeling/remodeling and OTM. We describe how the genetic factors in these important pathways may also influence external apical root resorption (EARR) concurrent with OTM. At the end of the chapter, known genetic factors in two conditions that could secondarily affect OTM as they increase treatment complexity (dental primary failure of eruption and dental agenesis) are reviewed, and a select group of syndromes and other genetic conditions that may affect OTM in patients are also summarized.