Leonard D. Kohn

Genomic DNA Released by Dying Cells Induces the Maturation of APCs

Mature APCs play a key role in the induction of Ag-specific immunity. This work examines whether genomic DNA released by dying cells provides a stimulus for APC maturation. Double-stranded but not single-stranded genomic DNA triggered APC to up-regulate expression of MHC class I/II and various costimulatory molecules. Functionally, dsDNA enhanced APC function in vitro and improved primary cellular and humoral immune responses in vivo. These effects were dependent on the length and concentration of the dsDNA but were independent of nucleotide sequence. The maturation of APC induced by dsDNA may promote host survival by improving immune surveillance at sites of tissue injury/infection.

The Thyrotropin Receptor

This chapter has outlined the complex process required for thyroid growth and function. Both events are regulated by TSHR via a multiplicity of signals, with the aid of and requirement for a multiplicity of hormones that regulate the TSHR via receptor cross-talk: insulin, IGF-I, adrenergic receptors, and purinergic receptors. Cross-talk appears to regulate G-protein interactions or activities induced by TSH as well as TSHR gene expression. The TSHR structure and its mechanism of signal transduction is being rapidly unraveled in several laboratories, since the recent cloning of the receptor. In addition, the epitopes for autoantibodies against the receptor that can subvert the normal regulated synthesis and secretion of thyroid hormones, causing hyper- or hypofunction, have been defined. Studies of regulation of the TSHR minimal promotor have uncovered a better understanding of the mechanisms by which TSH regulates both growth and function of the thyroid cell. A key novel component of this phenomenon involves TSH AMP positive and negative regulation of the TSHR. Negative transcriptional regulation is a common feature of MHC class I genes in the thyroid. Subversion of negative regulation or too little negative regulation is suggested to result in autoimmune disease. Methimazole and iodide at autoregulatory levels may be important in reversing this process and returning thyroid function to normal. Their action appears to involve factors that react with the IREs on both the TSHR and the TG promoter. Too much negative regulation, as in the case of ras transformation, results in abnormal growth without function. TTF-1 is implicated as a critical autoregulatory component in both positive and negative regulation of the TSHR and appears to be the link between TSH, the TSHR, TSHR-mediated signals, TG and TPO biosynthesis, and thyroid hormone formation. Differentially regulated expression of the TSHR and TG by cAMP and insulin depend on differences in the specificity of the TTF-1 site, that is, the lack of Pax-8 interactions with the TSHR, and the IRE sites. Single-strand binding proteins will become important in determining how TSHR transcription is controlled mechanistically.

Interaction of Hepatitis C Virus-Like Particles and Cells: a Model System for Studying Viral Binding and Entry

ABSTRACT Hepatitis C virus-like particles (HCV-LPs) containing the structural proteins of HCV H77 strain (1a genotype) was used as a model for HCV virion to study virus-cell interaction. HCV-LPs showed a buoyant density of 1.17 to 1.22 g/cm3 in a sucrose gradient and formed double-shelled particles 35 to 49 nm in diameter. Flow cytometry analysis by an indirect method (detection with anti-E2 antibody) and a direct method (use of dye-labeled HCV-LPs) showed that HCV-LPs binds to several human hepatic (primary hepatocytes, HepG2, HuH7, and NKNT-3) and T-cell (Molt-4) lines. HCV-LPs binding to cells occurred in a dose- and calcium-dependent manner and was not mediated by CD81. Scatchard plot analysis suggests the presence of two binding sites for HCV-LPs with high (Kd ∼1 μg/ml) and low (Kd ∼50 to 60 μg/ml) affinities of binding. Anti-E1 and -E2 antibodies inhibited HCV-LPs binding to cells. While preincubation of HCV-LPs with very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), or high-density lipoprotein (HDL) blocked its binding to cells, preincubation of cells with VLDL, LDL, HDL, or anti-LDL-R antibody did not. Confocal microscopy analysis showed that, after binding to cells, dye-labeled HCV-LPs were internalized into the cytoplasm. This process could be inhibited with anti-E1 or anti-E2 antibodies, suggesting that E1 and E2 proteins mediate HCV-LPs binding and, subsequently, their entry into cells. Altogether, our results indicate that HCV-LPs can be used to further characterize the mechanisms involved in the early steps of HCV infection.

Role of the Asialoglycoprotein Receptor in Binding and Entry of Hepatitis C Virus Structural Proteins in Cultured Human Hepatocytes

ABSTRACT We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.

Expression of Hypothalamic–Pituitary–Thyroid Axis Related Genes in the Human Skin

The skin is commonly affected in thyroid diseases, but the mechanism for this association is still unclear. As the skin expresses numerous neuroendocrine elements, we tested the additional cutaneous expression of mediators operating in the hypothalamic-pituitary-thyroid axis. We found significant expression of the thyroid-stimulating hormone receptor mRNA in cultured keratinocytes, epidermal melanocytes, and melanoma cells. The presence of thyroid-stimulating hormone receptor was confirmed by northern analyses and the thyroid-stimulating hormone receptor was found to be functionally active in cyclic adenosine monophosphate signal assays. Thyroid-stimulating hormone receptor expressing cells also expressed the sodium iodide symporter and thyroglobulin genes. We also found expression of deiodinases 2 and 3 (mainly deiodinase 2) in whole skin biopsy specimens, and in the majority of epidermal and dermal cells by reverse transcription-polymerase chain reaction followed by sequencing of the amplified gene segments. There was selective expression of the gene for thyroid-stimulating hormone beta; detection of the thyroid-releasing hormone gene was minimal and thyroid-releasing hormone receptor mRNA was not detected in most of the samples. Expression of functional thyroid-stimulating hormone receptor in the skin may have significant physiologic and pathologic consequences, particularly in autoimmune conditions associated with production of stimulating antibodies against the thyroid-stimulating hormone receptor. We conclude that the expanding list of neuroendocrine elements expressed in the skin supports a strong role for this system in cutaneous biology.

Wnt5a is expressed in murine and human atherosclerotic lesions

Atherosclerosis is an inflammatory disease involving the accumulation of macrophages in the intima. Wnt5a is a noncanonical member of the Wnt family of secreted glycoproteins. Recently, human macrophages have been shown to express Wnt5a upon stimulation with bacterial pathogens in vitro and in granulomatous lesions in the lung of Mycobacterium tuberculosis-infected patients. Wnt5a expression has also been liked to Toll-like receptor-4 (TLR-4), an innate immune receptor implicated in atherosclerosis. These observations, along with the fact that Wnt5a is involved in cell migration and proliferation, led us to postulate that Wnt5a plays a role in atherosclerosis. To investigate this hypothesis, we characterized Wnt5a expression in murine and human atherosclerotic lesions. Tissue sections derived from the aortic sinus to the aortic arch of apolipoprotein E-deficient mice and sections derived from the carotid arteries of patients undergoing endarterectomy were subjected to immunohistochemical analysis. All samples were found to be positive for Wnt5a with predominant staining in the areas of macrophage accumulation within the intima. In parallel, we probed for the presence of TLR-4 and found coincident TLR-4 and Wnt5a expression. For both the Wnt5a and TLR-4 staining, consecutive tissue sections treated with an isotype- and species-matched Ig served as a negative control and exhibited little, if any, reactivity. Quantitative RT-PCR revealed that Wnt5a mRNA expression in RAW264.7 murine macrophages can be induced by stimulation with LPS, a known ligand for TLR-4. Combined, these findings demonstrate for the first time Wnt5a expression in human and murine atherosclerotic lesions and suggest that cross talk between TLR-4 and Wnt5a is operative in atherosclerosis.

Thyroid Transcription Factor 1 Is Calcium Modulated and Coordinately Regulates Genes Involved in Calcium Homeostasis in C Cells

ABSTRACT Thyroid transcription factor 1 (TTF-1) was identified for its critical role in thyroid-specific gene expression; its level in the thyroid is regulated by thyrotropin-increased cyclic AMP levels. TTF-1 was subsequently found in lung tissue, where it regulates surfactant expression, and in certain neural tissues, where its function is unknown. Ligands or signals regulating TTF-1 levels in lung or neural tissue are unknown. We recently identified TTF-1 in rat parafollicular C cells and parathyroid cells. In this report, we show that TTF-1 is present in the parafollicular C cells of multiple species and that it interacts with specific elements on the 5′-flanking regions of the extracellular Ca2+-sensing receptor (CaSR), calmodulin, and calcitonin genes in C cells. When intracellular Ca2+ levels are increased or decreased in C cells, by the calcium ionophore A23187, by physiologic concentrations of the P2 purinergic receptor ligand ATP, or by changes in extracellular Ca2+ levels, the promoter activity, RNA levels, and binding of TTF-1 to these genes are, respectively, decreased or increased. The changes in TTF-1 inversely alter CaSR gene and calcitonin gene expression. We show, therefore, that TTF-1 is a Ca2+-modulated transcription factor that coordinately regulates the activity of genes critical for Ca2+homeostasis by parafollicular C cells. We hypothesize that TTF-1 similarly coordinates Ca2+-dependent gene expression in all cells in which TTF-1 and the CaSR are expressed, i.e., parathyroid cells, neural cells in the anterior pituitary or hippocampus, and keratinocytes.

Sequence similarity between cholera toxin and glycoprotein hormones: Implications for structure activity relationship and mechanism of action

Abstract The B chain of cholera toxin and the β subunits of thyrotropin, luteinizing hormone, human chorionic gonadotropin, and follicle-stimulating hormone are shown to have a region of sequence analogy believed to correlate with their ability to bind to receptors on cell membranes. A possible sequence analogy is also defined in the α subunits of these glycoprotein hormones and a region of the cholera toxin A 1 chain believed to be responsible for adenylate cyclase activation.

Graves’ IgG stimulation of continuously cultured rat thyroid cells: a sensitive and potentially useful clinical assay

A continuously cultured line of normal rat thyroid (FRTL) cells can be stimulated by immunoglobulin preparations from patients with Graves’ disease as measured by increases in intracellular cAMP levels. Responsiveness is concentration-dependent but is delayed in time relative to thyrotropin. Additionally, the cells respond to Graves’ immunoglobulins which have no long-acting thyroid stimulator (LATS) activity and are negative when adenylate cyclase stimulation in human thyroid membrane preparations is assayed. No correlation exists between the stimulation activity and the ability of a Graves’ immunoglobulin preparation to inhibit thyrotropin binding; cells are responsive even in the presence of such inhibitor activity.

Cholera toxin inhibits interferon action

Abstract Cholera toxin added together with interferon inhibited the establishment of antiviral activity in mouse L-cells. Cholera toxin has no effect on antiviral activity established before its addition; however, treatment of cells with the toxin before addition of interferon also inhibited the establishment of antiviral activity.