Serkan Sertel

SAMHD1 restricts HIV-1 infection in resting CD4 + T cells

Unlike activated CD4+ T cells, resting CD4+ T cells are highly resistant to productive HIV-1 infection. Early after HIV-1 entry, a major block limits reverse transcription of incoming viral genomes. Here we show that the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 prevents reverse transcription of HIV-1 RNA in resting CD4+ T cells. SAMHD1 is abundantly expressed in resting CD4+ T cells circulating in peripheral blood and residing in lymphoid organs. The early restriction to infection in unstimulated CD4+ T cells is overcome by HIV-1 or HIV-2 virions into which viral Vpx is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx-mediated proteasomal degradation of SAMHD1 and elevation of intracellular deoxynucleotide pools precede successful infection by Vpx-carrying HIV. Resting CD4+ T cells from healthy donors following SAMHD1 silencing or from a patient with Aicardi-Goutières syndrome homozygous for a nonsense mutation in SAMHD1 were permissive for HIV-1 infection. Thus, SAMHD1 imposes an effective restriction to HIV-1 infection in the large pool of noncycling CD4+ T cells in vivo. Bypassing SAMHD1 was insufficient for the release of viral progeny, implicating other barriers at later stages of HIV replication. Together, these findings may unveil new ways to interfere with the immune evasion and T cell immunopathology of pandemic HIV-1.

In vivo expression profile of the antiviral restriction factor and tumor-targeting antigen CD317/BST-2/HM1.24/tetherin in humans

Human CD317 is an intrinsic immunity factor that restricts the release of enveloped viruses, including the major pathogens HIV and Lassa virus, from infected cells in culture. Its importance for infection control in humans is unclear, due in part to its incompletely defined in vivo expression pattern. CD317 also has been proposed as a selective target for immunotherapy of multiple myeloma. To provide a framework for studies of the biological functions, regulation, and therapeutic potential of CD317, we performed microarray-based expression profiling in 468 tissue samples from 25 healthy organs from more than 210 patients. We found that CD317 protein was expressed to varying degrees in all organs tested and detected in a number of specialized cell types, including hepatocytes, pneumocytes, ducts of major salivary glands, pancreas and kidney, Paneth cells, epithelia, Leydig cells, plasma cells, bone marrow stromal cells, monocytes, and vascular endothelium. Although many of these cell types are in vivo targets for pathogenic viruses, restriction by CD317 or virus-encoded antagonists has been documented in only some of them. Limited cell type–dependent coexpression of CD317 with the IFN biomarker MxA in vivo and lack of responsive stimulation in organ explants suggest that interferons may only partially regulate CD317. This in vivo expression profiling sheds light on the biology and species-specificity of CD317, identifies multiple thus far unknown interaction sites of viruses with this restriction factor, and refutes the concept of its restricted constitutive expression and primary IFN inducibility. CD317s widespread expression calls into question its suitability as a target for immunotherapy.

Induction of CD4 + T-cell anergy and apoptosis by activated human B cells

B cells are well-known mediators of humoral immunity and serve as costimulators in the generation of T cell-mediated responses. In several mouse models, however, it was observed that B cells can also down-regulate immune reactions, suggesting a dual role for B cells. Due to this discrepancy and so far limited data, we directly tested the effects of primary human B cells on activated CD4(+) T helper cells in vitro. We found that under optimal costimulation large, activated CD25(+) B cells but not small CD25(-) B cells induced temporary T-cell anergy, determined by cell division arrest and down-regulation of cytokine production. In addition, large CD25(+) B cells directly induced CD95-independent apoptosis in a subpopulation of activated T cells. Suppression required direct B-T-cell contact and was not transferable from T to T cell, excluding potential involvement of regulatory T cells. Moreover, inhibitory effects involved an IL-2-dependent mechanism, since decreasing concentrations of IL-2 led to a shift from inhibitory toward costimulatory effects triggered by B cells. We conclude that activated CD25(+) B cells are able to costimulate or down-regulate T-cell responses, depending on activation status and environmental conditions that might also influence their pathophysiological impact.

Factors determining sensitivity or resistance of tumor cell lines towards artesunate

Clinical oncology is still challenged by the development of drug resistance of tumors that result in poor prognosis for patients. There is an urgent necessity to understand the molecular mechanisms of resistance and to develop novel therapy strategies. Artesunate (ART) is an anti-malarial drug, which also exerts profound cytotoxic activity towards cancer cells. We first applied a gene-hunting approach using cluster and COMPARE analyses of microarray-based transcriptome-wide mRNA expression profiles. Among the genes identified by this approach were genes from diverse functional groups such as structural constituents of ribosomes (RPL6, RPL7, RPS12, RPS15A), kinases (CABC1, CCT2, RPL41), transcriptional and translational regulators (SFRS2, TUFM, ZBTB4), signal transducers (FLNA), control of cell growth and proliferation (RPS6), angiogenesis promoting factors (ITGB1), and others (SLC25A19, NCKAP1, BST1, DBH, FZD7, NACA, MTHFD2). Furthermore, we applied a candidate gene approach and tested the role of resistance mechanisms towards established anti-cancer drugs for ART resistance. By using transfected or knockout cell models we found that the tumor suppressor p16(INK4A) and the anti-oxidant protein, catalase, conferred resistance towards ART, while the oncogene HPV-E6 conferred sensitivity towards ART. The tumor suppressor p53 and its downstream protein, p21, as well as the anti-oxidant manganese-dependent superoxide dismutase did not affect cellular response to ART. In conclusion, our pharmacogenomic approach revealed that response of tumor cells towards ART is multi-factorial and is determined by gene expression associated with either ART sensitivity or resistance. At least some of the functional groups of genes (e.g. angiogenesis promoting factors, cell growth and proliferation-associated genes signal transducers and kinases) are also implicated in clinical responsiveness of tumors towards chemotherapy. It merits further investigation, whether ART is responsive in clinically refractory tumors and whether the genes identified in the present study also determine clinical responsiveness towards ART.

SAMHD1’s protein expression profile in humans

The deoxynucleoside triphosphate triphosphohydrolase and 3′ → 5′ exonuclease SAMHD1 restricts HIV‐1 infection in noncycling hematopoietic cells in vitro, and SAMHD1 mutations are associated with AGS. Little is known about the in vivo expression and functional regulation of this cellular factor. Here, we first assessed the SAMHD1 protein expression profile on a microarray of 25 human tissues from >210 donors and in purified primary cell populations. In vivo, SAMHD1 was expressed in the majority of nucleated cells of hematopoietic origin, including tissue‐resident macrophages, DCs, pDCs, all developmental stages of thymic T cells, monocytes, NK cells, as well as at lower levels in B cells. Of note, SAMHD1 was abundantly expressed in HIV target cells residing in the anogenital mucosa, providing a basis for its evaluation as a cellular factor that may impact the efficiency of HIV transmission. Next, we examined the effect of the activation status and proinflammatory cytokine treatment of cells on expression and phosphorylation of SAMHD1. Activated, HIV‐susceptible CD4+ T cells carried pSAMHD1(T592), whereas resting CD4+ T cells and macrophages expressed the unphosphorylated protein with HIV‐restrictive activity. Surprisingly, stimulation of these primary cells with IFN‐α, IFN‐γ, IL‐4, IL‐6, IL‐12, IL‐18, IL‐27, or TNF‐α affected neither SAMHD1 expression levels nor threonine 592 phosphorylation. Only IL‐1β moderately down‐regulated SAMHD1 in activated CD4+ T cells. Taken together, this study establishes the first cross‐sectional protein expression profile of SAMHD1 in human tissues and provides insight into its cell cycle‐dependent phosphorylation and unresponsiveness to multiple proinflammatory cytokines.

Molecular docking and pharmacogenomics of vinca alkaloids and their monomeric precursors, vindoline and catharanthine.

Vinblastine and vincristine are dimeric indole alkaloids derived from Catharanthus roseus (formerly: Vinca rosea). Their monomeric precursor molecules are vindoline and catharanthine. While vinblastine and vincristine are well-known mitotic spindle poisons, not much is known about vindoline and catharanthine. Vindoline and catharanthine showed weak cytotoxicity, while vinblastine, vincristine, and the semisynthetic vindesine and vinorelbine revealed high cytotoxicity towards cancer cells. This may reflect a general biological principle of poisonous plants. Highly toxic compounds are not only active towards predators, but also towards plant tissues. Hence, plants need mechanisms to protect themselves from their own poisons. One evolutionary strategy to solve this problem is to generate less toxic precursors, which are dimerized to toxic end products when needed. As shown by in silico molecular docking and biochemical approaches, vinblastine, vincristine and vinorelbine bound with high affinity to α/β-tubulin and inhibited tubulin polymerization, whereas the effects of vindoline and catharanthine were weak. Similarly, vinblastine produced high fractions of mono- and multipolar mitotic spindles, while vindoline and catharanthine did only weakly affect bipolar mitotic spindle formation. Here, we show that vinblastine contributes to cell death by interference with spindle polarity. P-glycoprotein-overexpressing multidrug-resistant CEM/VCR1000 cells were highly resistant towards vincristine and cross-resistant to vinblastine, vindesine, and vinorelbine, but not or only weakly cross-resistant to vindoline and catharanthine. In addition to tubulin as primary target, microarray-based mRNA signatures of responsiveness of these compounds have been identified by COMPARE and signaling pathway profiling.

Determinants in HIV-1 Nef for enhancement of virus replication and depletion of CD4+ T lymphocytes in human lymphoid tissue ex vivo.

BackgroundHIV-1 Nef critically contributes to AIDS in part by augmenting virus titers in infected individuals. Analyzing which of Nefs activities contribute to HIV pathogenesis has been hampered by the lack of a cell culture model in which Nef exerts pronounced effects on HIV replication. The human lymphoid aggregate culture (HLAC) from tonsil maintains the cell populations and cytokine milieu found in vivo, supports a productive infection without exogenous stimulation, and Nef contributes to efficient HIV-1 replication as well as CD4+ T cell depletion in this experimental ex vivo-model.ResultsTo identify determinants in Nef that mediate these activities, we infected HLAC with a panel of isogenic HIV-1NL4-3 strains that encode for well-characterized mutants of HIV-1SF2 Nef. Determination of HIV-1 replication revealed that enhancement of the virus spread by Nef is governed by a complex set of protein interaction surfaces. In contrast, increased CD4+ T lymphocyte depletion depended on only two protein interaction surfaces in Nef that mediate either downregulation of cell surface CD4 or interaction with the NAKC signalosome. Consistently, in HLAC from 9 out of 14 donors, Nef enhanced CD4+ T cell depletion in the absence of a significant effect on virus replication. Moreover, our results suggest that this Nef-dependent enhancement in depletion occurred predominately in uninfected bystander CD4+ T cells.ConclusionOur findings suggest that Nef facilitates depletion of CD4+ T lymphocytes in HIV-1-infected lymphoid tissue ex vivo by increasing the pool of productively infected cells and by sensitizing bystander cells for killing. This ability might contribute to Nefs pathogenic potential in vivo.

Acupuncture for nasal congestion: a prospective, randomized, double-blind, placebo-controlled clinical pilot study.

Background Nasal congestion is one of the most common complaints dealt with in otorhinolaryngology. Side effects of decongestants are frequently seen in patients with chronic nasal congestion. This leads to an increasing demand of alternative treatments such as acupuncture. Future studies on acupuncture should aim at objectifying effects by both physical measuring and double blinding. Therefore, we were interested in whether these effects can potentially be measured as increase in nasal airflow (NAF) in ventus (“wind”) disease of traditional Chinese medicine (TCM). Methods Twenty-four patients with a history of nasal congestion due to hypertrophic inferior turbinates or chronic sinusitis without polyposis were additionally diagnosed according to the Heidelberg model of TCM. They were asked to score the severity of their nasal congestion on a visual analog scale (VAS). The acupuncturist was blinded according to the Heidelberg blinding assay. NAF was measured by using active anterior rhinomanometry (ARM). Specific verum acupoints according to the Chinese medical diagnosis were tested against nonspecific control acupoints. VAS and NAF were scored and measured before and 15 and 30 minutes after acupuncture. Results Control acupuncture showed a significant improvement in VAS and a deterioration of NAF. Verum acupuncture showed highly significant improvements in VAS and NAF. In addition, verum acupuncture improved NAF and VAS significantly over time. Conclusion Our control and verum acupoints fulfill the condition of a control and verum treatment, respectively. Measuring NAF by RRM and scoring VAS are possible and reflect acupuncture effects in vivo.

Anticancer activity of Salvia officinalis essential oil against HNSCC cell line (UMSCC1)

BACKGROUND Every year there are several hundred thousand new cases of oral cancer worldwide. Clinical oncology is still challenged by toxicity and side effects of multimodal therapy strategies in which is associated with poor prognosis for patients. There is an urgent necessity to develop novel therapy strategies. As the majority of anticancer drugs are of natural origin, natural products represent a valuable source for the identification and development of novel treatment options for cancer. The aim of this investigation was to study the cytotoxicity of Salvia officinalis L. (sage) essential oil. METHODS Salvia officinalis essential oil was gained by aqueous extraction from plant material and subsequently analyzed by gas chromatography. The cytotoxicity of the essential oil on the squamous human cell carcinoma cell line of the oral cavity (UMSCC1) was assessed with the XTT assay. These experiments revealed the half maximal inhibitory concentration (IC(50)) of the essential oil. It was used in the microarray-based analysis of gene expression of UMSSC1 cells. The results were submitted to a signaling pathway analysis. RESULTS The main constituents of Salvia officinalis essential oil include the monoterpenes thujone, β-pinene, and 1,8-cineol. Low concentrations of the essential oil increased vitality of the UMSCC1 cells. Beyond the concentration of the IC(50) of 135 µg/ml, sage essential oil reduced UMSSC1 cells viability to a minimum. In the microarray gene expression analysis, genes involved in cancer, cellular growth and proliferation, cell death, cell morphology, cell cycle, gene expression, and DNA repair were the most prominent. The three most significantly regulated pathways by sage were aryl hydrocarbon receptor signaling, cell cycle (G1/S checkpoint) regulation, and p53 signaling. CONCLUSION To the best of our knowledge, this study suggests for the first time the ability of Salvia officinalis essential oil to inhibit human HNSCC cell growth. The therapeutic potential of sage essential oil might exceed that of its common use in otorhinolaryngology.

Krebshemmende Wirkung des ätherischen Salbei-Öls auf eine Plattenepithelzellkarzinom-Zelllinie der Mundhöhle (UMSCC1)

BACKGROUND Every year there are several hundred thousand new cases of oral cancer worldwide. Clinical oncology is still challenged by toxicity and side effects of multimodal therapy strategies in which is associated with poor prognosis for patients. There is an urgent necessity to develop novel therapy strategies. As the majority of anticancer drugs are of natural origin, natural products represent a valuable source for the identification and development of novel treatment options for cancer. The aim of this investigation was to study the cytotoxicity of Salvia officinalis L. (sage) essential oil. METHODS Salvia officinalis essential oil was gained by aqueous extraction from plant material and subsequently analyzed by gas chromatography. The cytotoxicity of the essential oil on the squamous human cell carcinoma cell line of the oral cavity (UMSCC1) was assessed with the XTT assay. These experiments revealed the half maximal inhibitory concentration (IC(50)) of the essential oil. It was used in the microarray-based analysis of gene expression of UMSSC1 cells. The results were submitted to a signaling pathway analysis. RESULTS The main constituents of Salvia officinalis essential oil include the monoterpenes thujone, β-pinene, and 1,8-cineol. Low concentrations of the essential oil increased vitality of the UMSCC1 cells. Beyond the concentration of the IC(50) of 135 µg/ml, sage essential oil reduced UMSSC1 cells viability to a minimum. In the microarray gene expression analysis, genes involved in cancer, cellular growth and proliferation, cell death, cell morphology, cell cycle, gene expression, and DNA repair were the most prominent. The three most significantly regulated pathways by sage were aryl hydrocarbon receptor signaling, cell cycle (G1/S checkpoint) regulation, and p53 signaling. CONCLUSION To the best of our knowledge, this study suggests for the first time the ability of Salvia officinalis essential oil to inhibit human HNSCC cell growth. The therapeutic potential of sage essential oil might exceed that of its common use in otorhinolaryngology.