Lymphoma models for B-cell activation and tolerance. II. Growth inhibition by anti-mu of WEHI-231 and the selection and properties of resistant mutants

Regulation of the growth of murine B-cell lymphomas has been used as a model for tolerance induction. The inhibition by anti-immunoglobulin reagents of the growth of WEHI-231 and several variant clones has now been studied. The parental line is exquisitely sensitive to growth inhibition by heterologous or monoclonal anti-mu or anti-k reagents and ceases to incorporate thymidine within 24-48 hr of exposure to anti-immunoglobulin reagents. Growth inhibition is initially reversible, but prolonged exposure to anti-mu results in cell death. This inhibition is specific for immunoglobulin light and heavy chains since growth is not inhibited by antibodies directed at either class I or class II histocompatibility antigens. In order to study the mechanism of growth inhibition, we have mutagenized WEHI-231 with ethylmethane sulfonate and cloned the surviving colonies in the presence of anti-mu. Such variants, which have been repeatedly recloned, are able to grow normally in the presence of anti-mu up to 100 micrograms/ml. These "resistant" clones, while expressing amounts of surface IgM similar to that observed on WEHI-231, do not differ markedly in their ability to cap their immunoglobulin receptors compared to the parental line but appear to have lost class II antigens. Cell cycle analysis revealed that anti-mu causes a block in the transition of WEHI-231 from G1 to S phase. The relevance of these processes to models of B-cell tolerance induction are discussed.     

Inhibition of renin release following left circumflex bradykinin injection in dogs

We examined the renin secretory response to bradykinin (BK) injection into the left circumflex coronary artery (LCx) in dogs. Studies were conducted in anesthetized, carotid sinus denervated dogs which had been maintained on a low sodium diet. A 25 ga needle was inserted into the LCx for injection of BK (0.15 micrograms/kg). The rate of renin secretion (RS) was obtained during a 30 min control period, at 5 min after a non-hypotensive hemorrhage (10 ml/kg), at 1, 3 and 5 min after BK injection and at 15 min after the reinfusion of withdrawn blood. Four series of studies were conducted. Series I: BK injection into the LCx, Series II: saline injection into the LCx (sham), Series III: intravenous injection of BK, and Series IV: BK injection into the LCx in dogs with prior renal denervation. RS was suppressed by 80% (P less than 0.05) 5 min after injection of BK into the LCx. Saline injection (sham) into the LCx or intravenous BK administration did not inhibit RS. Furthermore, suppression of RS was not present in dogs with prior renal denervation. These results indicate that BK injection into the LCx causes a prompt reduction in the rate of RS and that this response is reflexively mediated by the renal nerves.     

Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells

Background

The incidence of papillary thyroid carcinoma (PTC) has risen steadily over the past few decades as well as the recurrence rates. It has been proposed that targeted ablative physical therapy could be a therapeutic modality in thyroid cancer. Targeted bio-affinity functionalized multi-walled carbon nanotubes (BioNanofluid) act locally, to efficiently convert external light energy to heat thereby specifically killing cancer cells. This may represent a promising new cancer therapeutic modality, advancing beyond conventional laser ablation and other nanoparticle approaches.

Methods

Thyroid Stimulating Hormone Receptor (TSHR) was selected as a target for PTC cells, due to its wide expression. Either TSHR antibodies or Thyrogen or purified TSH (Thyrotropin) were chemically conjugated to our functionalized Bionanofluid. A diode laser system (532 nm) was used to illuminate a PTC cell line for set exposure times. Cell death was assessed using Trypan Blue staining.

Results

TSHR-targeted BioNanofluids were capable of selectively ablating BCPAP, a TSHR-positive PTC cell line, while not TSHR-null NSC-34 cells. We determined that a 2:1 BCPAP cell:α-TSHR-BioNanofluid conjugate ratio and a 30 second laser exposure killed approximately 60% of the BCPAP cells, while 65% and >70% of cells were ablated using Thyrotropin- and Thyrogen-BioNanofluid conjugates, respectively. Furthermore, minimal non-targeted killing was observed using selective controls.

Conclusion

A BioNanofluid platform offering a potential therapeutic path for papillary thyroid cancer has been investigated, with our in vitro results suggesting the development of a potent and rapid method of selective cancer cell killing. Therefore, BioNanofluid treatment emphasizes the need for new technology to treat patients with local recurrence and metastatic disease who are currently undergoing either re-operative neck explorations, repeated administration of radioactive iodine and as a last resort external beam radiation or chemotherapy, with fewer side effects and improved quality of life.     

A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin

BACKGROUND: Cell transfection requires cationic DNA complexes and heparan sulfate proteoglycans (HSPGs) at the cell surface. Syndecans are transmembrane HSPGs that are ubiquitously expressed on adherent cells. Their polyanionic heparan sulfate moieties are bound at the distal end of their ectodomain, thus facilitating interaction with large cationic particles. METHODS: We propose a model for cell entry involving syndecans as receptors for the DNA complexes by comparing transfection with bacteria uptake and using drug inhibition experiments along with confocal microscopy. RESULTS: When combined with results from the literature, our data suggest the following sequence of events: after initial particle binding, gradual electrostatic zippering of the plasma membrane onto the particle is sustained by lateral diffusion of syndecan molecules that cluster into cholesterol-rich rafts. Clustering in turn triggers PKC activity and linker protein-mediated actin binding to the cytoplasmic tail of the syndecans. Resulting tension fibers and a growing network of cortical actin may then pull the particle into the cell. CONCLUSIONS: Diversion of integrin- and syndecan-mediated cell adhesion processes for particle engulfment appears to be widely exploited by animals (chylomicrons), by pathogens (bacteria, viruses) and, as suggested here, by non-viral vectors.     

Vav1 and Ly-GDI two regulators of Rho GTPases,function cooperatively as signal transducers in T cell antigen receptor-induced pathways

The Rho family GTPases are pivotal for T cell signaling; however, the regulation of these proteins is not fully known. One well studied regulator of Rho GTPases is Vav1; a hematopoietic cell-specific guanine nucleotide exchange factor critical for signaling in T cells, including stimulation of the nuclear factor of activated T cells (NFAT). Surprisingly, Vav1 associates with Ly-GDI, a hematopoietic cell-specific guanine nucleotide dissociation inhibitor of Rac. Here, we studied the functional significance of the interaction between Vav1 and Ly-GDI in T cells. Upon organization of the immunological synapse, both Ly-GDI and Vav1 relocalize to T cell extensions in contact with the antigen-presenting cell. Ly-GDI is phosphorylated on tyrosine residues following T cell receptor stimulation, and it associates with the Src homology 2 region of an adapter protein, Shc. In addition, the interaction between Ly-GDI and Vav1 requires tyrosine phosphorylation. Overexpression of Ly-GDI alone is inhibitory to NFAT stimulation and calcium mobilization. However, when co-expressed with Vav1, Ly-GDI enhances Vav1 induction of NFAT activation, phospholipase Cgamma phosphorylation, and calcium mobilization. Moreover, Ly-GDI does not alter the regulation of these phenomena when coexpressed with oncogenic Vav1. Since oncogenic Vav1 does not bind Ly-GDI, this suggests that the functional cooperativity of Ly-GDI and Vav1 is dependent upon their association. Thus, our data suggest that the interaction of Vav1 and Ly-GDI creates a fine tuning mechanism for the regulation of intracellular signaling pathways leading to NFAT stimulation.     

Id2 negatively regulates B cell differentiation in the spleen

Early stages of B cell development occur in the bone marrow, resulting in formation of immature B cells. These immature cells migrate to the spleen where they differentiate into mature (B2 or marginal zone (MZ)) cells. This final maturation step is crucial for B cells to become responsive to Ags and to participate in the immune response. Id2 is a helix-loop-helix protein that lacks a DNA-binding region; and therefore, inhibits basic helix-loop-helix functions in a dominant negative manner. In this study, we show that Id2 expression is down-regulated during differentiation of immature B cells into mature B2 and MZ B cells. The high levels of Id2 expressed in the immature B cells result in inhibition of E2A binding activity to an E2 box site. Moreover, mice lacking Id2 show an elevation in the proportion of mature B2 cells in the spleen, while the MZ population in these mice is almost absent. Thus, Id2 acts as a regulator of the differentiation of immature B cells occurring in the spleen, it negatively controls differentiation into mature B2 cells while allowing the commitment to MZ B cells. In the absence of Id2 control, the unregulated differentiation is directed toward the mature B2 population.     

Preferential Th1 immune response in invariant chain-deficient mice

MHC class II molecules associate with the invariant chain (Ii) molecule during biosynthesis. Ii facilitates the folding of class II molecules, interferes with their peptide association, and is involved in MHC class II transport. In this study, we have investigated the in vitro and in vivo immune response of Ii-deficient mice (Ii(-/-)). Our results have demonstrated that CD4(+) T cells from Ii(-/-) mice proliferate normally in vitro after in vivo immunization with protein Ags. However, cytokine secretion profiles of Ag-primed CD4(+) T cells from Ii(-/-) mice differ from CD4(+) T cells from wild-type mice. Whereas cells from wild-type mice secrete IFN-gamma and IL-4, cells from Ii(-/-) mice secrete mostly IFN-gamma. Moreover, Ii(-/-) mice exhibit a normal Th1 response in the delayed-type hypersensitivity and trinitrobenzene sulfonic acid colitis models; however, these mice lack an in vivo Th2 response, as demonstrated in the asthma model. Therefore, we suggest that defective Ag presentation in Ii(-/-) mice leads selectively to a Th1 effector response.