FGF23-Associated Tumor-Induced Osteomalacia in a Patient With Small Cell Carcinoma: A Case Report and Regulatory Mechanism Study.

Tumor-induced osteomalacia (TIO) is typically caused by phosphaturic mesenchymal tumor (PMT) that secretes the phosphaturic hormone, fibroblast growth factor-23 (FGF23), resulting in decreased phosphate reabsorption in kidneys, hypophosphatemia, and finally osteomalacia. Rare cases of malignant tumor manifesting with TIO other than PMT had been reported, although in most of these reports, except one, circulating FGF23 levels were not evaluated and tissue expressing of FGF23 was not confirmed. In this article, we report a case of TIO in a patient with pulmonary small cell carcinoma with liver metastasis. The patient manifested with hypophosphatemia. His circulating level of FGF23 was markedly increased. The expression of FGF23 in tumor cells was confirmed. Furthermore, the regulatory mechanism of FGF23 in this patient was also investigated.

Cell-cell fusion and internalization of the CNS-based, HIV-1 co-receptor, APJ.

APJ, a member of the human G protein-coupled seven-transmembrane receptor family, has been shown to serve as a coreceptor for the entry of human immunodeficiency virus type I (HIV-1) and simian immunodeficiency virus (SIV), and it is dramatically expressed in central nervous system (CNS)-based cells. In this study, expression of APJ tagged with the green fluorescent protein (GFP) and a fluorescent peptide, 5-carboxyfluorescein (5-CF) conjugated Apelin-13, were utilized for studying receptor internalization and recycling, in stably expressing indicator cells, human neurons, primary CNS microvascular endothelial cells (MVECs), and astrocytes. Fusion of the C-terminus of APJ to the N-terminus of GFP did not alter receptor ligand binding and functions, including signaling and internalization. Using 293 cells stably expressing APJ-GFP, we demonstrated that rapid internalization of the APJ receptor was induced by stimulation with Apelin-36 and Apelin-13, in a dose-dependent manner. Furthermore, investigations showed that the internalized APJ was colocalized with transferrin receptors, suggesting that the internalization of APJ induced by Apelin is likely to be via clathrin-coated pits. Interestingly, we found that the internalized APJ molecules were recycled to the cell surface within 60 min after removal of Apelin-13, but most of the internalized APJ still remained in the cytoplasm, even 2 h after washout of Apelin-36. The intact cytoplasmic C-terminal domain was found to be required for ligand-induced APJ internalization. Human neurons were dramatically stained by the APJ-binding fluorescent peptides. Primary human fetal astrocytes were less strongly labeled with 5-CF-Apelin-13, and in primary human CNS MVECs only weak distribution of green fluorescence specific for APJ in the cytoplasm was observed. Apelin-36 blocked cell membrane fusion mostly due to steric interference, with only a very modest effect on receptor internalization. The CNS represents a unique reservoir site for HIV-1. As such, molecular therapeutics and small molecular inhibitors of HIV-1 entry via this unique CNS receptor are now able to be rationally designed.

Potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (HIV-1) Vif proteins.

Virion infectivity factor (Vif) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) in vivo, but its function remains uncertain. Recently, we have shown that Vif proteins are able to form multimers, including dimers, trimers, or tetramers. Because the multimerization of Vif proteins is required for Vif function in the viral life cycle, we propose that it could be a novel target for anti-HIV-1 therapeutics. Through a phage peptide display method, we have identified a set of 12-mer peptides containing a PXP motif that binds to HIV-1 Vif protein. These proline-enriched peptides potently inhibited the Vif-Vif interaction in vitro. We have also screened a set of synthesized Vif peptides (15-mer), which covers all the amino acids of the HIV-1 Vif protein sequence, for their ability to inhibit the Vif-Vif interaction in vitro. We demonstrated that Vif-derived proline-enriched peptides that contain the (161)PPLP(164) domain are able to inhibit the Vif-Vif interaction. Conversely, the deletion of the (161)PPLP(164) domain of Vif protein will significantly impair the capability of Vif proteins to interact with each other, indicating that the (161)PPLP(164) domain plays a key role in Vif multimerization. All these results demonstrate that the proline-enriched peptides block the multimerization of Vif through interfering with the polyproline interfaces of Vif formed by (161)PPLP(164) domain. Moreover, these peptides which inhibit the Vif-Vif interaction in vitro potently inhibit HIV-1 replication in the "nonpermissive" T-cells. We propose that this study starts a novel strategy to develop structural diverse inhibitors of Vif such as peptidomimetics or small organic molecules.

Peripheral blood Dendritic cells are not a major reservoir for HIV type 1 in infected individuals on virally suppressive HAART.

Dendritic cells (DCs) are potent antigen-presenting cells, and their physiological localization in tissues that interact with the external environment is important as a first barrier against pathogens such as human immunodeficiency virus type I (HIV-1). Several models have been proposed to explain the possible role of DCs as a reservoir for HIV-1 in patients on virally suppressive highly active antiretroviral therapy (HAART). However, the low yield of cell isolates has made this evaluation a difficult task. The present study analyzes whether peripheral blood DCs from HIV-1-infected individuals on virally suppressive HAART, with plasma HIV-1 RNA levels of less than 50 copies/ml, carry either HIV-1 provirus and/or HIV-1 virions. Peripheral blood DCs were isolated from a cohort of 10 HIV-1-seropositive men taking suppressive HAART. In five patients, plasmacytoid and myeloid dendritic cells were isolated to attempt to identify their respective roles in HIV-1 residual disease. Viral out-growth assays were performed in vitro, as well as gag and R/U5 polymerase chain reaction (PCR) amplification of viral RNA and DNA, respectively, from DC and peripheral blood mononuclear cell (PBMC) extracts. Fluorescence activated cell-sorting (FACS) data revealed cellular yields from 85.90 to 95.18%, of relatively pure DCs isolated from patients' PBMCs. Although HIV-1 RNA gag and DNA RU/5 were detected in all PBMC samples isolated from the patients, proviral DNA and viral RNA forms were not detected in any of the DC isolates. In addition, no replication-competent virus was demonstrated in DC coculture assays, while virus was isolated from each patients' CD8+ T-lymphocyte-depleted PBMC cocultures. Furthermore, HIV-1 gag proviral DNA was not detected in either plasmacytoid or myeloid DC subfractions. The current study suggests that in HIV-1-infected individuals treated with suppressive HAART, peripheral blood DCs do not carry HIV-1 proviral DNA or viral particles attached to their surface. These populations of peripheral blood DCs are likely not a major HIV-1 reservoir in patients on HAART with clinically undetectable plasma viral RNA.

Ethanol strongly potentiates apoptosis induced by HIV-1 proteins in primary human brain microvascular endothelial cells.

Ethanol may have significant effects on human immunodeficiency virus type I (HIV-1) pathogenesis in vivo. As such, the effects of ethanol treatment were studied on the proapoptotic potential of various HIV-1 proteins in primary isolated human brain microvascular endothelial cells (MVECs), a major cellular component of the blood-brain barrier. Low-passage primary brain MVECs were treated with recombinant HIV-1 proteins Nef, Vpr, Tat and gp120 proteins from X4, R5, and X4R5 viral strains, with and without ethanol at various relevant concentrations. The apoptotic potential of each HIV-1 protein with and without ethanol was compared with cells treated with ethanol alone or GST protein as a control, under similar conditions. Specific HIV-1 proteins induced apoptosis in primary isolated human brain MVECs, which was potentiated on treatment with 0.1 and 0.3% (v/v) ethanol. Cotreatment with ethanol and specific HIV-1 proteins showed enhanced lactate dehydrogenase release, compared with MVECs treated with ethanol alone. The presence of ethanol in in vitro culture medium also enhanced HIV-1 protein-mediated tumor necrosis factor-alpha production, compared with cells treated with ethanol alone or GST protein. Thus, these studies demonstrate ethanol's potential for inducing apoptosis of human MVECs with relevant HIV-1-specific proteins and suggest a potential synergistic effect in augmenting HIV-1 neuroinvasion and neuropathogenesis in vivo.

Primary isolated human brain microvascular endothelial cells express diverse HIV/SIV-associated chemokine coreceptors and DC-SIGN and L-SIGN.

Chemokines have received increasing attention due to their inhibitory activities on human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV) replication and the potential for chemokine receptors to assist in HIV-1/SIV entry into permissive cells. Besides CD4, which is the major receptor for HIV-1 and SIV, a number of chemokine receptors including but not limited to APJ, CCR3, CXCR4, and CCR5 may be coreceptors for HIV-1/SIV, not only in peripheral blood and lymphoid tissues but also in the central nervous system (CNS). The present studies reveal the lack of CD4, but the significant expression of various chemokine receptors, APJ, CCR3, CXCR4, and CCR5, plus C-type lectins DC-SIGN and L-SIGN on isolated primary human brain microvascular endothelial cells (MVECs). As these MVECs do not express CD4, this suggests a CD4-independent HIV/SIV entry/infection of these cells, which are the major cells constituting the human blood-brain barrier. We also found that chemokines for cognate chemokine receptors individually were unable to block binding of HIV-1 to brain MVECs. These results reveal that in primary isolated brain MVECs viral attachment is mediated by a possible previously unknown receptor(s) or by cooperative activity of various receptors. Moreover, mRNA transcripts for DC-SIGN/L-SIGN, as well as DC-SIGN protein expression, suggest the capability of MVECs to attach viral particles on cell surfaces, even though polyclonal antisera for DC-SIGN did not affect viral binding to these cells. These data will assist in further understanding lentiviral entry into the CNS.

Lentiviral expression of HIV-1 Vpr induces apoptosis in human neurons.

Our recent studies have demonstrated that extracellular, recombinant human immunodeficiency virus type I (HIV-1) Vpr protein is highly neurotoxic in the microenvironment of differentiated mature human neurons and undifferentiated neuronal precursors. Although most of the direct neurotoxic effects of HIV-1 have been attributed previously to the envelope gene product, gp120, and the Tat regulatory protein, it was demonstrated that Vpr protein caused apoptosis comparable to that induced by gp120 protein in a dose-dependent manner in the neuronal system. Having observed the neurocytopathic effects of extracellular Vpr protein previously, the effects of virally expressed Vpr on nondividing, terminally differentiated human neurons were investigated. An HIV-1-based three-plasmid expression vector system was utilized to study the effects of intracellularly expressed Vpr. These virion preparations were then used to transduce neurons generated from the human neuronal precursor NT2 cell-line. Intracellularly expressed Vpr induced apoptosis within terminally differentiated neurons, as demonstrated by TUNEL assays. Additionally, virions lacking Vpr expression did not significantly induce apoptosis within these neurons. These results suggest that HIV-1 Vpr may also be leading directly to selective neurotoxicity through intracellular expression. Furthermore, human apoptosis gene microarray comparisons exhibited an up-regulation of Bcl-2-related mRNA, as well as other apoptosis genes involved in the mitochondrial apoptotic pathway, for the Vpr-transduced neuronal cells, when compared to Vpr-negative controls. Thus, Vpr delivered intracellularly, as well as extracellularly, is involved in the induction of significant neuronal apoptosis and may be one of the molecular mechanisms in HIV-1-induced encephalopathy.