Toll-Like Receptors 7, 8, and 9 Expression and Function in Primary Human Cervical Cancer Langerhans Cells Evidence of Anergy

Objective: Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. Methodology: Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a(+)CD207(+) cells. Acute monocytic leukemia cell line THP-1-derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1 beta, IL-6, IL-10, IL-12p70, interferon (IFN) alpha, interferon gamma, and tumor necrosis factor (TNF) alpha by Luminex multiplex bead array. Human papillomavirus was genotyped. Results: We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs. expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1 beta, and tumor necrosis factor alpha to TLR8 ligand and interferon alpha in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. Conclusions: Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

Methionine down-regulates TLR4/MyD88/NF-kappa B signalling in osteoclast precursors to reduce bone loss during osteoporosis

Background and PurposeStudies have demonstrated that a moderate intake of amino acids is associated with development of bone health. Methionine, a sulphur-containing essential amino acid, has been largely implicated for improving cartilage formation, however its physiological significance on bone integrity and functionality have not been elucidated. We investigated whether methionine can prevent osteoporotic bone loss. Experimental ApproachThe anti-resorptive effect of methionine, (250mgkg(-1) body wt administered in drinking water for 10 weeks), was evaluated in ovariectomized (OVX) rats by monitoring changes in bone turnover, formation of osteoclasts from blood-derived mononuclear cells and changes in the synthesis of pro-osteoclastogenic cytokines. Key resultsMethionine improved bone density and significantly decreased the degree of osteoclast development from blood mononuclear cells in OVX rats, as indicated by decreased production of osteoclast markers tartarate resistant acid phosphatase b (TRAP5b) and MIP-1. siRNA-mediated knockdown of myeloid differentiation primary response 88 MyD88], a signalling molecule in the toll-like receptor (TLR) signalling cascade, abolished the synthesis of both TRAP5b and MIP-1 in developing osteoclasts. Methionine supplementation disrupted osteoclast development by inhibiting TLR-4/MyD88/NF-B pathway. Conclusions and ImplicationsTLR-4/MyD88/NF-B signalling pathway is integral for osteoclast development and this is down-regulated in osteoporotic system on methionine treatment. Methionine treatment could be beneficial for the treatment of postmenopausal osteoporosis.

The Glycosylated Rv1860 Protein of Mycobacterium tuberculosis Inhibits Dendritic Cell Mediated TH1 and TH17 Polarization of T Cells and Abrogates Protective Immunity Conferred by BCG

We previously reported interferon gamma secretion by human CD4(+) and CD8(+) T cells in response to recombinant E. coli-expressed Rv1860 protein of Mycobacterium tuberculosis (MTB) as well as protection of guinea pigs against a challenge with virulent MTB following prime-boost immunization with DNA vaccine and poxvirus expressing Rv1860. In contrast, a Statens Serum Institute Mycobacterium bovis BCG (BCG-SSI) recombinant expressing MTB Rv1860 (BCG-TB1860) showed loss of protective ability compared to the parent BCG strain expressing the control GFP protein (BCG-GFP). Since Rv1860 is a secreted mannosylated protein of MTB and BCG, we investigated the effect of BCG-TB1860 on innate immunity. Relative to BCG-GFP, BCG-TB1860 effected a significant near total reduction both in secretion of cytokines IL-2, IL-12p40, IL-12p70, TNF-alpha, IL-6 and IL-10, and up regulation of co-stimulatory molecules MHC-II, CD40, CD54, CD80 and CD86 by infected bone marrow derived dendritic cells (BMDC), while leaving secreted levels of TGF-beta unchanged. These effects were mimicked by BCG-TB1860His which carried a 6-Histidine tag at the C-terminus of Rv1860, killed sonicated preparations of BCG-TB1860 and purified H37Rv-derived Rv1860 glycoprotein added to BCG-GFP, but not by E. coli-expressed recombinant Rv1860. Most importantly, BMDC exposed to BCG-TB1860 failed to polarize allogeneic as well as syngeneic T cells to secrete IFN-gamma and IL-17 relative to BCG-GFP. Splenocytes from mice infected with BCG-SSI showed significantly less proliferation and secretion of IL-2, IFN-gamma and IL-17, but secreted higher levels of IL-10 in response to in vitro restimulation with BCG-TB1860 compared to BCG-GFP. Spleens from mice infected with BCG-TB1860 also harboured significantly fewer DC expressing MHC-II, IL-12, IL-2 and TNF-alpha compared to mice infected with BCG-GFP. Glycoproteins of MTB, through their deleterious effects on DC may thus contribute to suppress the generation of a TH1- and TH17-dominated adaptive immune response that is vital for protection against tuberculosis.

Characterization of type I interferon pathway during hepatic differentiation of human pluripotent stem cells and hepatitis C virus infection

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-alpha and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-alpha treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs - LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival. (C) 2015 The Authors. Published by Elsevier B.V.

The secretion of gonadotrophin-releasing hormone by peri-implantation embryos of the rhesus monkey: comparison with the secretion of chorionic gonadotrophin

Chorionic gonadotrophin (CG) is the first clear embryonic signal during early pregnancy in primates. CG has close structural and functional similarities to pituitary luteinizing hormone (LH) which is regulated by gonadotrophin releasing hormone (GnRH). To study the regulatory mechanism of CG secretion in primate embryos, we examined the production and timing of secretion of GnRH in peri-implantation embryos of the rhesus monkey. In-vivo fertilized/developed morulae and early blastocysts, recovered from non-superovulated, naturally-bred rhesus monkeys by non-surgical uterine flushing, were cultured in vitro to hatched, attached and post-attached blastocyst stages using a well-established culture system. We measured GnRH and CG in media samples from cultured embryos with a sensitive radioimmunoassay and bioassay, respectively. The secretion of GnRH (pg/ml; mean +/- SEM) by embryos (n = 20) commenced from low levels (0.32 +/- 0.05) during the pre-hatching blastocyst stage to 0.70 +/- 0.08 at 6-12 days and 1.30 +/- 0.23 at greater than or equal to 13 days of hatched blastocyst attachment and proliferation of trophoblast cells. GnRH concentrations in culture media obtained from embryos (n = 5) that failed to hatch and attach were mostly undetectable (less than or equal to 0.1). Samples that did not contain detectable GnRH failed to show detectable CG. Immunocytochemical studies, using a specific monoclonal anti-GnRH antibody (HU4H) as well as polyclonal antisera (LR-1), revealed that immunopositive GnRH cells were localized in pre-hatching blastocysts (n = 4), in blastocysts (n = 2) after 5-10 days of attachment and in monolayer cultures (n = 4) of well-established embryonic trophoblast cells. GnRH positive staining was seen only in cytotrophoblasts but not in syncytiotrophoblasts. Similarly, cytotrophoblast, but not syncytiotrophoblast, cells of the rhesus placenta were immunopositive. In controls, either in the absence of antibody or in the presence of antibody pre-absorbed with GnRH, these cells failed to show stain. These observations indicate, for the first time, that an immunoreactive GnRH is produced and secreted by blastocysts during the peri-attachment period and by embryo-derived cytotrophoblast cells in the rhesus monkey.

Gene expression profile of epithelial cells and mesenchymal cells derived from limbal explant culture

Purpose: Limbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.Methods: RNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4x44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction. Results: The microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.Conclusions: This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

Patterned growth and differentiation of neural cells on polymer derived carbon substrates with micro/nano structures in vitro

The growth of neuroblastoma (N2a) and Schwann cells has been explored on polymer derived carbon substrates of varying micro and nanoscale geometries: resorcinol-formaldehyde (RE) gel derived carbon films and electrospun nanofibrous (similar to 200 nm diameter) mat and SU-8 (a negative photoresist) derived carbon micro-patterns. MTT assay and complementary lactate dehydrogenase (LDH) assay established cytocompatibility of RE derived carbon films and fibers over a period of 6 days in culture. The role of length scale of surface patterns in eliciting lineage-specific adaptive response along, across and on the interspacing between adjacent micropatterns (i.e., ``on'', ``across'' and ``off'') has been assayed. Textural features were found to affect 3',5'-cyclic AMP sodium salt-induced neurite outgrowth, over a wide range of length scales: from similar to 200 nm (carbon fibers) to similar to 60 mu m (carbon patterns). Despite their innate randomness, carbon nanofibers promoted preferential differentiation of N2a cells into neuronal lineage, similar to ordered micro-patterns. Our results, for the first time, conclusively demonstrate the potential of RE-gel and SU-8 derived carbon substrates as nerve tissue engineering platforms for guided proliferation and differentiation of neural cells in vitro. (C) 2013 Elsevier Ltd. All rights reserved.

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H2O2, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl(+) cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl(+) CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl(+) cells. NAC enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.

Intracellular activities of Salmonella enterica in murine dendritic cells.

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.

Synthesis, Characterization and Evaluation of Cytotoxicity of New Aminophosphonic Acid Diesters in Human Leukemia Cells

A series of novel fluoroaminophosphates 4a-4j were synthesized by one-pot method in presence of tetramethylguanidine (TMG) as a catalyst and were characterized by elemental analysis, FTIR, H-1, C-13, P-31, F-19 NMR, and mass spectra. All the title compounds were evaluated forin vitro cytotoxicity against leukemic cell line derived from T-cells of leukemia patient (CEM cells) by Trypan blue exclusion and MTT assays, and these were found to exert concentration dependent cytotoxic effects. Among them 4f, 4g & 4j possessed marked cytotoxicity. 4g (with IC50 value of 6 mu M) had emerged as lead compound.

Expression of cyclin E in endomitotic silk-gland cells from mulberry silkworm

The silk glands of mulberry silkworm Bombyx mori are endoreplicating tissues in which the genomic DNA undergoes multiple rounds of replication without mitosis and nuclear division. In the absence of normal mitotic division, the cell cycle essentially alternates between the G1 and S phases. Cyclin E is crucial for the G1/S transition in both mitotic and endoreplicating cycles. We have cloned and characterized cyclin E (cyclin box) from B. mori, which is nearly identical to the Drosophila cyclin E box except for an insertion of 21 amino acids. Two distinct cyclin E transcripts (1.7 and 2.1 kb) were detected in the silk-gland cells of B. mori and in the B. mori-derived embryonic cell line, BmN. Using anti Cyclin E antibodies two protein bands of 52 and 44 kDa were detected in silk glands and BmN cells at Comparable levels. Both BmN- and the silk-gland cells showed the presence of the interacting kinase Cdk2. Transcripts of the mitotic cyclin, cyclin B, were barely detectable in the endoreplicating silk-gland cells and amounted to only 4-7% of that seen in the mitotically dividing BmN cells. The near absence of cyclin B transcripts and the abundant expression of cyclin E in the silk glands correlate well with the alternation of only G1 and S phases without the intervening mitosis in these cells. (C) 2000 Elsevier Science B.V. All rights reserved.

Studies on nonisothermal solid state galvanic cells — effect of gradients on EMF

An expression for the EMF of a nonisothermal galvanic cell, with gradients in both temperature and chemical potential across a solid electrolyte, is derived based on the phenomenological equations of irreversible thermodynamics. The EMF of the nonisothermal cell can be written as a sum of the contributions from the chemical potential gradient and the EMF of a thermocell operating in the same temperature gradient but at unit activity of the neutral form of the migrating species. The validity of the derived equation is confirmed experimentally by imposing nonlinear gradients of temperature and chemical potential across galvanic cells constructed using fully stabilized zirconia as the electrolyte. The nature of the gradient has no effect on the EMF.

Functional Identification of Regulatory Elements within the Promoter Region of Platelet-Derived Growth Factor 2

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2,the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure.In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.

In vitro protection of umbilical cord blood-derived primitive hematopoietic stem progenitor cell pool by mannose-specific lectins via antioxidant mechanisms.

BACKGROUND: Earlier we reported that an oral administration of two mannose-specific dietary lectins, banana lectin (BL) and garlic lectin (GL), led to an enhancement of hematopoietic stem and progenitor cell (HSPC) pool in mice. STUDY DESIGN AND METHODS: Cord blood derived CD34+ HSPCs were incubated with BL, GL, Dolichos lectin (DL), or artocarpin lectin (AL) for various time periods in a serum- and growth factor free medium and were subjected to various functional assays. Reactive oxygen species (ROS) levels were detected by using DCHFDA method. Cell fractionation was carried out using lectin-coupled paramagnetic beads. RESULTS: CD34+ cells incubated with the lectins for 10 days gave rise to a significantly higher number of colonies compared to the controls, indicating that all four lectins possessed the capacity to protect HSPCs in vitro. Comparative analyses showed that the protective ability of BL and GL was better than AL and DL and, therefore, further experiments were carried out with them. The output of long-term culture-initiating cell (LTC-IC) and extended LTC-IC assays indicated that both BL and GL protected primitive stem cells up to 30 days. The cells incubated with BL or GL showed a substantial reduction in the ROS levels, indicating that these lectins protect the HSPCs via antioxidant mechanisms. The mononuclear cell fraction isolated by lectin-coupled beads got enriched for primitive HSPCs, as reflected in the output of phenotypic and functional assays.CONCLUSION: The data show that both BL and GL protect the primitive HSPCs in vitro and may also serve as cost-effective HSPC enrichment tools.