Kedar Shrestha

Modification of surfactant protein D by reactive oxygen-nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo

Surfactant protein D (SP‐D) is an important effector of innate immunity. We have previously shown that SP‐D accumulates at sites of acute bacterial infection and neutrophil infiltration, a setting associated with the release of reactive species such as peroxynitrite. Incubation of native SP‐D or trimeric SP‐D lectin domains (NCRDs) with peroxynitrite resulted in nitration and nondisulfide cross‐linking. Modifications were blocked by peroxynitrite scavengers or pH inactivation of peroxynitrite, and mass spectroscopy confirmed nitration of conserved tyrosine residues within the C‐terminal neck and lectin domains. Mutant NCRDs lacking one or more of the tyrosines allowed us to demonstrate preferential nitration of Tyr314 and the formation of Tyr228‐dependent cross‐links. Although there was no effect of peroxynitrite or tyrosine mutations on lectin activity, incubation of SP‐D dodecamers or murine lavage with peroxynitrite decreased the SP‐D‐dependent aggregation of lipopolysaccharide‐coated beads, supporting our hypothesis that defective aggregation results from abnormal cross‐linking. We also observed nitration, cross‐linking of SP‐D, and a significant decrease inSP‐D‐dependent aggregating activity in the lavage of mice acutely exposed to nitrogen dioxide. Thus, modification of SP‐D by reactive oxygen‐nitrogen species could contribute to alterations in the structure and function of SP‐D at sites of inflammation in vivo.— Matalon, S., Shrestha, K., Kirk, M., Waldheuser, S., McDonald, B., Smith, K., Gao, Z., Belaaouaj, A., Crouch, E. C. Modification of surfactant protein D by reactive oxygen‐nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo. FASEB J. 23, 1415–1430 (2009)

Inhibition of Na+ Transport in Lung Epithelial Cells by Respiratory Syncytial Virus Infection

We investigated the mechanisms by which respiratory syncytial virus (RSV) infection decreases vectorial Na+ transport across respiratory epithelial cells. Mouse tracheal epithelial (MTE) cells from either BALB/c or C57BL/6 mice and human airway H441 cells were grown on semipermeable supports under an air-liquid interface. Cells were infected with RSV-A2 and mounted in Ussing chambers for measurements of short-circuit currents (I(sc)). Infection with RSV for 24 hours (multiplicity of infection = 1) resulted in positive immunofluorescence for RSV antigen in less than 10% of MTE or H441 cells. In spite of the limited number of cells infected, RSV reduced both basal and amiloride-sensitive I(sc) in both MTE and H441 cells by approximately 50%, without causing a concomitant reduction in transepithelial resistance. Agents that increased intracellular cAMP (forskolin, cpt-CAMP, and IBMX) increased mainly Cl(-) secretion in MTE cells and Na+ absorption in H441 cells. RSV infection for 24 hours blunted both variables. In contrast, ouabain sensitive I(sc), measured across apically permeabilized H441 monolayers, remained unchanged. Western blot analysis of H441 cell lysates demonstrated reductions in alpha- but not gamma-ENaC subunit protein levels at 24 hours after RSV infection. The reduction in amiloride-sensitive I(sc) in H441 cells was prevented by pretreatment with inhibitors of de novo pyrimidine or purine synthesis (A77-1726 and 6-MP, respectively, 50 microM). Our results suggest that infection of both murine and human respiratory epithelial cells with RSV inhibits vectorial Na+ transport via nucleotide release. These findings are consistent with our previous studies showing reduced alveolar fluid clearance after RSV infection of BALB/c mice.

The 5′‐untranslated RNA of the human dhfr minor transcript alters transcription pre‐initiation complex assembly at the major (core) promoter

The human dhfr minor transcript is distinguished from the predominant dhfr mRNA by an ∼400 nucleotide extension of the 5′‐untranslated region, which corresponds to the major (core) promoter DNA (its template). Based on its unusual sequence composition, we hypothesized that the minor transcript 5′‐UTR might be capable of altering transcription pre‐initiation complex assembly at the core promoter, through direct interactions of the RNA with specific regulatory polypeptides or the promoter DNA itself. We found that the minor transcript 5′‐UTR selectively sequesters transcription factor Sp3, and to a lesser extent Sp1, preventing their binding to the dhfr core promoter. This allows a third putative transcriptional regulatory protein, which is relatively resistant to sequestration by the minor transcript RNA, the opportunity to bind the dhfr core promoter. The selective sequestration of Sp3 > Sp1 by the minor transcript 5′‐UTR involves an altered conformation of the RNA, and a structural domain of the protein distinct from that required for binding to DNA. As a consequence, the minor transcript 5′‐UTR inhibits transcription from the core promoter in vitro (in trans) in a concentration‐dependent manner. These results suggest that the dhfr minor transcript may function in vivo (in cis) to regulate the transcriptional activity of the major (core) promoter.

Interregulation of Proton-gated Na+ Channel 3 and Cystic Fibrosis Transmembrane Conductance Regulator

Proton-gated Na+ channels (ASIC) are new members of the epithelial sodium channel/degenerin gene family. ASIC3 mRNA has been detected in the homogenate of pulmonary tissues. However, whether ASIC3 is expressed in the apical membranes of lung epithelial cells and whether it regulates cystic fibrosis transmembrane conductance regulator (CFTR) function are not known at the present time. Using reverse transcription-PCR, we found that the ASIC3 mRNA was expressed in the human airway mucosal gland (Calu-3) and human airway epithelial (16HBE14o) cells. Indirect immunofluorescence microscopy revealed that ASIC3 was co-segregated with CFTR in the apical membranes of Calu-3 cells. Proton-gated, amiloride-sensitive short circuit Na+ currents were recorded across Calu-3 monolayers mounted in an Ussing chamber. In whole-cell patch clamp studies, activation of CFTR channels with cAMP reduced proton-gated Na+ current in Calu-3 cells from -154 ± 28 to -33 ± 16 pA (n = 5, p < 0.05) at -100 mV. On the other hand, cAMP-activated CFTR activity was significantly inhibited following constitutive activation of putative ASIC3 at pH 6.0. Immunoassays showed that both ASIC3 and CFTR proteins were expressed and co-immunoprecipitated mutually in Calu-3 cells. Similar results were obtained in human embryonic kidney 293T cells following transient co-transfection of ASIC3 and CFTR. Our results indicate that putative CFTR and ASIC3 channels functionally interact with each other, possibly via an intermolecular association. Because acidic luminal fluid in the cystic fibrosis airway and lung tends to stimulate ASIC3 channel expression and activity, the interaction of ASIC3 and CFTR may contribute to defective salt and fluid transepithelial transport in the cystic fibrotic pulmonary system.

Oncogenes, Malignant Transformation, and Modern Medicine

During the past decade there have been remarkable strides in the understanding of the basic mechanism of cancer. It is now clear that there is a set of genes, known as oncogenes, that can cause cells to become malignant if their expression is altered, either by mutation or overexpression. The products of these genes include growth factors, growth factor receptors, signal tranduction proteins, and DNA binding proteins. The normal cellular counterparts of these genes play very important roles in the regulation of growth and proliferation by normal cells. Another set of genes, anti-oncogenes, also play an important role in preventing abnormal cell proliferation. The remarkable explosion of understanding of the pathophysiology of malignancy has led to a common unifying concept of malignant transformation that applies to all tumors. It is likely that these new insights will lead to improved and more specific treatments for malignant disease in the next decade.

Establishment of growth factor-dependent MOPC 104E cell line in vitro

The MOPC 104E cell line has been adapted to grow in vitro using a combination of feeder layer and growth factor(s). The growth of this myeloma cell line is dependent on the presence of growth factor(s). Growth-promoting activity generated from T-cell-mitogen-stimulated, Corynebacterium parvum-stimulated spleen cell culture supernatant, and peritoneal adherent cell culture supernatants gives dose-dependent proliferation. Generation of growth factors in the serum-free bovine serum albumin-substituted media and a rapid assay system based on [3H]thymidine uptake for the quantitation of growth promoting activity are described.

Characterization of 5B12.1, A Monoclonal Antibody Specific for IL-6

A monoclonal antibody (MAb) specific for interleukin-6 (IL-6) was generated by fusing SP2/0 cells with spleen cells from a mouse immunized with rat spleen cell derived plasmacytoma growth factor (rat PCT-GF). This MAb inhibited the growth of an IL-6-sensitive murine plasmacytoma clone, MD90, in the presence of the immunogen, rat PCT-GF. More interesting, however, this MAb demonstrated species cross-reactivity by neutralizing murine (recombinant and P388D1 cell line-derived) and human (recombinant) IL-6. IL-6 neutralization activity was also established in other IL-6 bioassays, such as the proliferation of spleen cells, plasmacytoma T1165, and a B-cell hybridoma 7TD1. IL-6 neutralization was overcome partially by increasing the concentration of PCT-GF. The MAb had no effect on PCT-GF-independent plasmacytoma KI81 proliferation. Plastic petri dish-bound MAb removed rmIL-6 activity. These results suggest that this MAb specifically binds IL-6 and neutralizes bioactivity of various PCT-GF, rmIL-6, and rhIL-6.

IRES inhibition induces terminal differentiation and synchronized death in triple-negative breast cancer and glioblastoma cells

Internal ribosome entry site (IRES)-mediated translation is a specialized mode of protein synthesis which malignant cells depend on to survive adverse microenvironmental conditions. Our lab recently reported the identification of a group of compounds which selectively interfere with IRES-mediated translation, completely blocking de novo IGF1R synthesis, and differentially modulating synthesis of the two c-Myc isoforms. Here, we examine the phenotypic consequences of sustained IRES inhibition in human triple-negative breast carcinoma and glioblastoma cells. A sudden loss of viability affects the entire tumor cell population after ∼72-h continuous exposure to the lead compound. The extraordinarily steep dose-response relationship (Hill-Slope coefficients −15 to −35) and extensive physical connections established between the cells indicate that the cells respond to IRES inhibition collectively as a population rather than as individual cells. Prior to death, the treated cells exhibit prominent features of terminal differentiation, with marked gains in cytoskeletal organization, planar polarity, and formation of tight junctions or neuronal processes. In addition to IGF1R and Myc, specific changes in connexin 43, BiP, CHOP, p21, and p27 also correlate with phenotypic outcome. This unusual mode of tumor cell death is absolutely dependent on exceeding a critical threshold in cell density, suggesting that a quorum-sensing mechanism may be operative. Death of putative tumor stem cells visualized in situ helps to explain the inability of tumor cells to recover and repopulate once the compound is removed. Together, these findings support the concept that IRES-mediated translation is of fundamental importance to maintenance of the undifferentiated phenotype and survival of undifferentiated malignant cells.

Immunoregulation of Murine Plasmacytoma I. Generation of Anomalous Killer Cells in Vitro by Cocultivation with MOPC 104E

Murine plasmacytoma MOPC 104E-K181 is a tissue culture cell line of MOPC 104E derived from BALB/c mice. MOPC 104E-K181 implanted subcutaneously in syngeneic normal mice regresses spontaneously after an initial growth of about 10 mm. Mice that regressed tumors or mice immunized intraperitoneally with mitomycin C-treated MOPC 104E-K181 myeloma could reject subsequent challenge of viable K181 myeloma cells. In contrast to euthymic mice, T-cell-deficient athymic nude mice developed subcutaneous tumors after challenge and died from progressive tumor growth, suggesting the critical role of T cells in tumor regression. In vitro induction of cytotoxic cells was used to define the immunologic mechanism by which the host can suppress tumor growth. Spleen cells from immune mice did not show cytolytic activity in 51Cr release cytotoxicity assay, but showed inhibitory action of tumor proliferation in vitro at an effector cell to target cell ratio of 500:1 in a [3H]thymidine incorporation assay. To determine if cytotoxicity could be induced against MOPC 104E-K181 cells, in vitro sensitizing cultures were studied. We have demonstrated that normal BALB/c spleen cells became cytotoxic against MOPC 104E-K181 cells after 5 days cultivation with mitomycin C-treated stimulator cells at an optimal responder to stimulator cell ratio of 5:1. Treatment of anti-Thy-1.2 serum plus complement abolished cytotoxic activity of effector cells. Cytotoxic cells lysed not only MOPC 104E-K181 cells used for stimulation but also H-2k osteosarcoma cells. It was concluded that Thy-1.2-positive cytotoxic cells with nonspecific anomalous reactivity could be induced in murine plasmacytoma-stimulating cultures.

Immunoregulation of Murine Plasmacytoma II. Target Selectivity of Anomalous Killer Cells and Role of Immune T Cells for the Induction

Requirements for the induction of anomalous killer (AK) cells to syngeneic MOPC 104E-KI81 plasmacytoma, and their pattern of reaction were investigated. The AK cells are Thy-1.2 positive and were induced by cocultivation of normal BALB/c spleen cells for 5 days in vitro with mitomycin C-treated MOPC 104E-KI81 cells in RPMI 1640 medium supplemented with fetal calf serum and 2-mercaptoethanol. The pattern of reactivity was investigated by direct cytotoxicity and indirect cold target inhibition assays using a panel of target cells. The results from cold target inhibition show that anomalous killer cells react with various tumor targets in the same preferential order as the results shown in direct cytotoxicity assay, and indicates that the AK cell population may be detecting the same determinants expressed to different degrees on panels of target cells. Kinetic study showed that peak cytotoxic responses were observed on day 5 and day 4, for primary and secondary sensitizing cultures, respectively. Cytotoxicity was not generated when nude mouse spleen cells were used as responder cells, suggesting the essential role of T cells in the induction. Nylon wool-column-purified splenic T cells from mice primed in vivo with intraperitoneal injection of MMC-treated stimulator cells, but not from normal mice, were able to generate AK cells in vitro. These results indicate that generation of AK cells with NK-cell-like reactivity needs inductive factors provided by the immune reaction between sensitized T cells and stimulatory MOPC 104E-KI81 cells.