Propofol Related Infusion Syndrome: Ultrastructural Evidence for a Mitochondrial Disorder.

OBJECTIVE: The objective of this report of a fatal propofol-related infusion syndrome in a young adult was to present-to our knowledge for the first time-direct ultrastructural evidence for the central role of mitochondrial damage in the pathogenesis of this syndrome. DATA SOURCES: Histological and electron microscopical analysis of liver, skeletal, and heart muscle obtained by autopsy and blood obtained from patient. STUDY SELECTION: Case report. DATA EXTRACTION: In addition to conventional macroscopical and histological investigations, electron-microscopical analysis of myocardial- and skeletal muscle and liver tissue obtained at autopsy from a young man was performed in order to search for ultrastructural changes of mitochondria. Acylcarnitine concentrations of his blood were determined by ultra-high performance liquid chromatography mass spectrometry. DATA SYNTHESIS: A 19-year-old male was admitted with acute left-side hemiparesis. The patient was intubated, then propofol infusion started, and a craniotomy was performed to remove an intracerebral hematoma. In the postoperative period, the patient presented with elevated intracranial pressure and brain edema. After repeat surgery, the patient showed impaired systolic left ventricular function, increasing fever, anuria, hyperkalemia, and metabolic acidosis, and he finally expired. Electron microscopy revealed dark, electron dense amorphous structures associated with mitochondria in heart muscle and liver tissue obtained at autopsy. Peripheral blood analysis revealed increased levels of acetyl-, propionyl-, butyryl-, malonyl-, and valeryl-carnitine as an indicator for propofol-related infusion syndrome, as well as for propofol-mediated inhibition of free fatty acid uptake into mitochondria, affecting beta-oxidation. CONCLUSIONS: Electron dense bodies found in association with mitochondria in muscle and liver cells probably correspond to accumulation of free fatty acid provide direct morphological evidence for the mitochondrial damage in propofol-related infusion syndrome.

Guanine modification of inhibitory oligonucleotides potentiates their suppressive function.

Inhibitory TLR7 and/or TLR9 oligonucleotides (inhibitory oligonucleotide [INH-ODN]) are characterized by a phosphorothioate backbone and a CC(T)XXX3₋5GGG motif, respectively. INH-ODN 2088 is a prototypic member of this class of INH-ODN and acts as a TLR7 and TLR9 antagonist. It contains a G quadruple that leads to higher order structures by the formation of G tetrads. These structures are unfavorable for the prediction of their pharmacological and immunological behavior. We show in this study that modification of Gs within the G quadruple by 7-deaza-guanine or 7-deaza-2'-O-methyl-guanine avoids higher order structures and improves their inhibitory potential. Whereas TLR9-induced TNF-α secretion of bone marrow-derived macrophages and conventional dendritic cells was equally inhibited by INH-ODN 2088 and G-modified INH-ODNs such as INH-ODN 24888, TLR7-induced TNF-α release and TLR7- and TLR9-induced IL-12p40 release were significantly more impaired by G-modified INH-ODNs. Similarly, the IL-6 release of B cells from wild-type and autoimmune MRL/Mp-lpr/lpr mice was more efficiently impaired by G-modified INH-ODNs. Surprisingly, INH-ODN 2088 stimulated B cells to proliferate when used in higher doses. Finally, in vivo, in wild-type and autoimmune MRL/Mp-lpr/lpr mice, G-modified INH-ODN 24888 was significantly more efficient than unmodified INH-ODN 2088. In summary, G modification allows the development of INH-ODNs with superior inhibitory potency for inflammatory diseases with high medical need such as systemic lupus erythematosus.

Induction of tumor stem cell differentiation--novel strategy to overcome therapy resistance in gastric cancer.

PURPOSE: Metastases are a frequent finding in gastric cancer and are associated with poor prognosis. A recently discovered link between metabolic changes, differentiation, and therapy resistance due to tumor stem cells could depict a novel approach in cancer research and therapy. Phosphoglycerate kinase 1 (PGK1) is a metabolic enzyme and is known to be involved in enabling gastric cancer cells to be invasive and to disseminate. In this study, we investigated if PGK1 is a promising candidate in inducing stem cell differentiation in gastric cancer. MATERIALS AND METHODS: MKN45 gastric cancer cells were used due to their known cancer stem cell population, which is defined by the surface marker CD44. MKN45 cells were separated between CD44+ and CD44- cells and, in equal parts, incubated with shRNA anti-PGK1 using fluorescence-activated cell sorting (FACS) analysis; they were then injected into nude mice to evaluate their tumor growth behavior in vivo. Further, the invasive potential of gastric cancer cells was evaluated in vitro using the xCelligence analyzing system. RESULTS: CD44+ gastric cancer cells treated with and without shRNA anti-PGK1 were capable to cause tumor growth in vivo, whereas tumor growth in CD44+ cells treated with shRNA anti-PGK1 was considerably smaller in comparison with that in CD44+ cells without treatment. CD44- cells did not show any noticeable tumor growth in vivo. By targeting PGK1, the invasive potential of gastric cancer cells was impressively reduced in vitro. In all our cells, which were targeted with shRNA anti-PGK1, we did not find any change that is in accordance with the phenotype of the cells using FACS analysis. CONCLUSIONS: Our findings suggest that targeting the key metabolic enzyme PGK1 in gastric cancer cells may open a new chapter in cancer treatment, which is well worth for further exploration in combination with recent chemotherapy, and might be a promising possibility to overcome therapy resistance in gastric cancer.

Toll-like receptor (TLR) 3 immune modulation by unformulated small interfering RNA or DNA and the role of CD14 (in TLR-mediated effects).

The Toll-like receptors (TLRs) 3, 7, 8 and 9 stimulate innate immune responses upon recognizing pathogen-derived nucleic acids. TLR3 is located on the cell surface and in cellular endosomes and recognizes double-stranded viral RNA or the synthetic mimic poly rI:rC. Recently, unformulated small interfering RNA (siRNA) has been reported as ligand for surface-expressed murine TLR3. Blockage of TLR3 is achieved by single-stranded DNA. We confirm and expand the observation that poly rI:rC-mediated TLR3 immune activation is blocked in a sequence-, length-, backbone- and CpG-dependent manner. However, human TLR3 is not activated by siRNA, which may be the result of differences in the amino acid composition of the TLR3 loop 1 of mice and humans. Although CD14 was previously described as a co-receptor for murine TLR3 and other nucleic acid-recognizing TLRs, human CD14 acts only as co-receptor to human TLR9, but not TLR3, TLR7 or TLR8. We show that CD14 up-regulates the TLR9 immune response of A, B and C-class oligodeoxynucleotides but down-regulates the phosphoro-diester version of B-class oligodeoxynucleotides.

Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies to certain cellular macromolecules, such as the small nuclear ribonucleoprotein particles (snRNPs), which had been considered to be passive targets of the autoimmune response. SLE is also characterized by the increased expression of type I interferon (IFN), which appears to be associated with the development and severity of disease. Here, we show that specific, highly conserved RNA sequences within snRNPs can stimulate Toll-like receptors (TLRs) 7 and 8 as well as activate innate immune cells, such as plasmacytoid dendritic cells (pDCs), which respond by secreting high levels of type I IFN. SLE patient sera containing autoantibodies to snRNPs form immune complexes that are taken up through the Fc receptor gammaRII and efficiently stimulate pDCs to secrete type I IFNs. These results demonstrate that a prototype autoantigen, the snRNP, can directly stimulate innate immunity and suggest that autoantibodies against snRNP may initiate SLE by stimulating TLR7/8.

The TLR7/8 ligand resiquimod targets monocyte-derived dendritic cell differentiation via TLR8 and augments functional dendritic cell generation.

Imidazoquinolone compounds, such as resiquimod are Toll-like receptor (TLR) 7/8 ligands representing novel immune response modifiers undergoing clinical testing. Resiquimod has been reported to modulate conventional human monocyte-derived DC (moDC) differentiation, but the role of TLR7 and TLR8 is unclear. We directly dissected the TLR7- and TLR8-dependency by employing selective TLR7 ligands and resiquimod-coculture experiments with inhibitory oligonucleotides (iODN) suppressing TLR7, TLR7+8 or TLR7+8+9. Selective TLR7 ligands did not affect conventional moDC differentiation as analyzed by CD14/CD1a expression. iODN experiments confirmed that resiquimod's effects during DC differentiation were antagonized only with TLR8 iODNs. Direct comparison of resiquimod DC with TLR7- and control-DC revealed significantly higher T-cell costimulatory molecule and MHC class II expression. Resiquimod DC promoted significantly stronger allogeneic T-cell proliferation and stronger naïve CD4(+) T-cell proliferation. These results indicate the relevance of TLR8 for human monocyte-derived DC differentiation and maturation and may be relevant for clinical trials employing resiquimod.

Components of the ligand for a Ni++ reactive human T cell clone.

The major histocompatibility complex (MHC) restriction element for a human Ni(2+) reactive T cell, ANi-2.3, was identified as DR52c. A series of experiments established that the functional ligand for this T cell was a preformed complex of Ni(2+) bound to the combination of DR52c and a specific peptide that was generated in human and mouse B cells, but not in fibroblasts nor other antigen processing-deficient cells. In addition, ANi-2.3 recognition of this complex was dependent on His81 of the MHC beta chain, suggesting a role for this amino acid in Ni(2+) binding to MHC. We propose a general model for Ni(2+) recognition in which betaHis81 and two amino acids from the NH(2)-terminal part of the MHC bound peptide coordinate Ni(2+) which then interacts with some portion of the Valpha CDR1 or CDR2 region.

Sequences derived from self-RNA containing certain natural modifications act as suppressors of RNA-mediated inflammatory immune responses.

The ability of the host to distinguish between self and foreign nucleic acids is one of the critical factors contributing to the recognition of pathogens by Toll-like receptors (TLRs). Under certain circumstances, eukaryotic self-RNA may reach TLR-containing compartments allowing for self-recognition. Specific modifications were previously demonstrated to suppress immune activation when placed at several positions in an immune stimulatory RNA or silencing RNA (siRNA). However, we show that even a simple natural modification such as a single 2'-O-methylation at different nucleotide positions throughout a sequence derived from a self-RNA strongly interferes with TLR-mediated effects. Such a single modification can even have an inhibitory effect in vitro and in vivo when placed in a different than the immune stimulatory RNA strand acting as suppressive RNA. Several safeguard mechanisms appear to have evolved to avoid cellular TLR-mediated activation by self-RNAs that may under other circumstances result in inflammatory or autoimmune responses. This knowledge can be used to include as few as a single 2'-O-methyl modification at a specific position in a siRNA sense or anti-sense strand to avoid TLR immune effects.

Arenavirus Induced CCL5 Expression Causes NK Cell-Mediated Melanoma Regression.

Immune activation within the tumor microenvironment is one promising approach to induce tumor regression. Certain viruses including oncolytic viruses such as the herpes simplex virus (HSV) and non-oncolytic viruses such as the lymphocytic choriomeningitis virus (LCMV) are potent tools to induce tumor-specific immune activation. However, not all tumor types respond to viro- and/or immunotherapy and mechanisms accounting for such differences remain to be defined. In our current investigation, we used the non-cytopathic LCMV in different human melanoma models and found that melanoma cell lines produced high levels of CCL5 in response to immunotherapy. In vivo, robust CCL5 production in LCMV infected Ma-Mel-86a tumor bearing mice led to recruitment of NK cells and fast tumor regression. Lack of NK cells or CCL5 abolished the anti-tumoral effects of immunotherapy. In conclusion, we identified CCL5 and NK cell-mediated cytotoxicity as new factors influencing melanoma regression during virotherapy.

Immunostimulatory effects of three classes of CpG oligodeoxynucleotides on PBMC from HCV chronic carriers.

BACKGROUND: Chronic hepatitis C virus (HCV) infection results from weak or absent T cell responses. Pegylated-interferon-alpha (IFN-alpha) and ribavirin, the standard of care for chronic HCV, have numerous immune effects but are not potent T cell activators. A potent immune activator such as TLR9 agonist CpG oligodeoxynucleotide (CpG) may complement current treatment approaches. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from HCV chronic carriers who failed previous treatment and from healthy donors were incubated in vitro with the three main CpG classes (A, B or C), recombinant IFN-alpha-2b (IntronA) and/or ribavirin. Proliferation and cytokine secretion (IFN-alpha, IL-10 and IP-10) were evaluated. RESULTS: CpG induced proliferation and cytokine secretion in patterns expected for each CpG class with similar group means for HCV and healthy donors. IntronA and ribavirin, alone or together, had no detectable effects. IntronA and C-Class CpG together induced more IFN-alpha than CpG alone in most subjects. IFN-alpha secretion was proportional to the number of plasmacytoid dendritic cells in PBMC from healthy donors but not HCV donors in whom responses were highly heterogeneous. CONCLUSION: The strong immune stimulatory effect of CpG on PBMC isolated from treatment-failed HCV patients suggests possible utility alone or in combination with current HCV antiviral treatment.

Characterization of conserved viral leader RNA sequences that stimulate innate immunity through TLRs.

Viruses of the order Mononegavirales encompass life-threatening pathogens with single-stranded segmented or nonsegmented negative-strand RNA genomes. The RNA genomes are characterized by highly conserved sequences at the extreme untranslated 3' and 5' termini that are most important for virus infection and viral RNA synthetic processes. The 3' terminal genome regions of negative-strand viruses such as vesicular stomatitis virus, Sendai virus, or influenza virus contain a high number of conserved U and G nucleotides, and synthetic oligoribonucleotides encoding such sequences stimulate sequence-dependent cytokine responses via TLR7 and TLR8. Immune cells responding to such sequences include NK cells, NK/T cells, plasmacytoid, and myeloid dendritic cells, as well as monocytes and B cells. Strong Th1 and pro-inflammatory cytokine responses are also induced upon in vivo application of oligoribonucleotides. It appears possible that the presence of highly conserved untranslated terminal regions in the viral genome fulfilling fundamental functions for the viral replication may enable the host to induce directed innate immune defense mechanisms, by allowing pathogen detection through essential RNA regions that the virus cannot readily mutate.

Therapeutic applications of synthetic CpG oligodeoxynucleotides as TLR9 agonists for immune modulation.

Vertebrate toll-like receptors (TLRs) sense invading pathogens by recognizing bacterial and viral structures and, as a result, activate innate and adaptive immune responses. Ten human functional TLRs have been reported so far; three of these (TLR7, 8, and 9) are expressed in intracellular compartments and respond to single-stranded nucleic acids as natural ligands. The pathogen structure selectively recognized by TLR9 in bacterial or viral DNA was identified to be CpG dinucleotides in specific sequence contexts (CpG motifs). Short phosphorothioate-stabilized oligodeoxynucleotides (ODNs) containing such motifs are used as synthetic TLR9 agonists, and different classes of ODN TLR9 agonists have been identified with distinct immune modulatory profiles. The TLR9-mediated activation of the vertebrate immune system suggests using such TLR9 agonists as effective vaccine adjuvants for infectious disease, and for the treatment of cancer and asthma/allergy. Immune activation by CpG ODNs has been demonstrated to be beneficial in animal models as a vaccine adjuvant and for the treatment of a variety of viral, bacterial, and parasitic diseases. Antitumor activity of CpG ODNs has also been established in numerous mouse models. In clinical vaccine trials in healthy human volunteers or in immunocompromised HIV-infected patients, CpG ODNs strongly enhanced vaccination efficiency. Most encouraging results in the treatment of cancers have come from human phase I and II clinical trials using CpG ODNs as a tumor vaccine adjuvant, monotherapy, or in combination with chemotherapy. Therefore, CpG ODNs represent targeted immune modulatory drugs with a broad range of potential applications.

CpG motifs to modulate innate and adaptive immune responses.

The vertebrate adaptive and innate immune systems have evolved to protect the host from pathogen infections. To achieve this mission, the innate immune system developed particular receptors, termed "pattern recognition receptors" (PRRs). These PRRs selectively bind certain types of structures expressed by pathogens but in principal absent in vertebrates. One of the best understood receptors is the Toll-like receptor (TLR) 9 that recognizes CpG sequence motifs in bacterial and viral DNA. Different classes of short synthetic phosphorothioate-stabilized CpG oligodeoxynucleotides were developed and are currently in human clinical trials in the fields of infectious disease, cancer, and asthma/allergy.

TLR9 in health and disease.

Toll-like receptor 9 (TLR9) is specialized for the recognition of pathogenic nucleic acids. TLR9 is expressed in intracellular compartments where it responds specifically to pathogen DNA. Several factors contribute to the ability of TLR9 to discriminate between self and foreign DNA. Regulatory mechanisms of the innate and adaptive immune system exist that balance the immune responses mediated by TLR9. Short synthetic CpG oligodeoxynucleotides are used to induce controlled and directed TLR9-dependent stimulation and are effective immune modulators in preclinical and clinical studies. This review will summarize the interplay between TLR9-dependent opposing stimulatory and regulatory effects in innate and adaptive immunity.

Combination therapy including CpG oligodeoxynucleotides and entecavir induces early viral response and enhanced inhibition of viral replication in a woodchuck model of chronic hepadnaviral infection.

CpG oligodeoxynucleotides (ODNs) stimulate immune cells via TLR9 and are potentially useful immunomodulators for the treatment of chronic viral infections. In the present study, different classes of CpGs were tested for their capacities for innate immune activation and antiviral activities in the woodchuck model. A class P CpG ODN was found to stimulate interferon (IFN) production in woodchuck peripheral blood mononuclear cells (PBMCs) in vitro, and following subcutaneous administration in vivo, it was observed to induce IFN and MxA expression in woodchuck PBMCs. Combination treatment with CpG ODN and entecavir (ETV) led to effective suppression of the woodchuck hepatitis virus (WHV) load in the woodchucks, with early viral responses and inhibition of replication. The woodchuck hepatitis surface antigen (WHsAg) serum concentrations were strongly decreased by CpG and ETV together but not by either agent alone, indicating synergistic effects. However, viral control post-treatment was still transient, similar to that observed with ETV alone. Significantly elevated levels of serum aspartate aminotransferase (AST) but not of alanine aminotransferase (ALT) in some of the woodchucks receiving CpG ODN were noted, but these increases were resolved before the completion of treatment and were not associated with an elevated serum bilirubin level or coagulation disorders, suggesting the absence of a significant safety concern.

Impact of modifications of heterocyclic bases in CpG dinucleotides on their immune-modulatory activity.

Synthetic phosphorothioate oligodeoxynucleotides (ODN) bearing unmethylated CpG motifs can mimic the immune-stimulatory effects of bacterial DNA and are recognized by Toll-like receptor 9 (TLR9). Past studies have demonstrated that nucleotide modifications at positions at or near the CpG dinucleotides can severely affect immune modulation. However, the effect of nucleotide modifications to stimulate human leukocytes and the mechanism by which chemically modified CpG ODN induce this stimulation are not well understood. We investigated the effects of CpG deoxyguanosine substitutions on the signaling mediated by human TLR9 transfected into nonresponsive cells. ODN incorporating most of these substitutions stimulated detectable TLR9-dependent signaling, but this was markedly weaker than that induced by an unmodified CpG ODN. One of the most active ODN tested contained deoxyinosine for deoxyguanosine substitutions (CpI ODN), but its relative activity to induce cytokine secretion on mouse cells was much weaker than on human cells. The activity was dependent on TLR9, as splenocytes from mice genetically deficient in TLR9 did not respond to CpI ODN stimulation. It is surprising that CpI ODN were nearly as strong as CpG ODN for induction of human B cell stimulation but were inferior to CpG ODN in their ability to induce T helper cell type 1 effects. These data indicate that certain deoxyguanosine substitutions in CpG dinucleotides are tolerated to stimulate a TLR9-mediated immune response, but this response is insufficient to induce optimal interferon-alpha-mediated effects, which depend on the presence of an unmodified CpG dinucleotide. These studies provide a structure-activity relationship for TLR9 agonist compounds with diverse immune effects.

A subset of human plasmacytoid dendritic cells expresses CD8α upon exposure to herpes simplex virus type 1.

Classical and plasmacytoid dendritic cells (DC) play important roles in the defense against murine and human infections with herpes simplex virus (HSV). So far, CD8α expression has only been reported for murine DC. CD8α(+) DC have prominent cross-presenting activities, which are enhanced by murine CD8α(+) PDC. The human orthologue of murine CD8α(+) DC, the CD141 (BDCA3)(+) DC, mainly cross-present after TLR3 ligation. We report here the serendipitous finding that a subset of human PDC upregulates CD8α upon HSV-1 stimulation, as shown by gene array and flow cytometry analyses. CD8α, not CD8ß, was expressed upon exposure. Markers of activation, migration, and costimulation were upregulated on CD8α-expressing human PDC. In these cells, increased cytokine and chemokine levels were detected that enhance development and function of T, B, and NK cells, and recruit immature DC, monocytes, and Th1 cells, respectively. Altogether, human CD8α(+) PDC exhibit a highly activated phenotype and appear to recruit other immune cells to the site of inflammation. Further studies will show whether CD8α-expressing PDC contribute to antigen cross-presentation, which may be important for immune defenses against HSV infections in vitro and in vivo.

Guanine-modified inhibitory oligonucleotides efficiently impair TLR7- and TLR9-mediated immune responses of human immune cells.

Activation of TLR7 and TLR9 by endogenous RNA- or DNA-containing ligands, respectively, is thought to contribute to the complicated pathophysiology of systemic lupus erythematosus (SLE). These ligands induce the release of type-I interferons by plasmacytoid dendritic cells and autoreactive antibodies by B-cells, both responses being key events in perpetuating SLE. We recently described the development of inhibitory oligonucleotides (INH-ODN), which are characterized by a phosphorothioate backbone, a CC(T)XXX3-5GGG motif and a chemical modification of the G-quartet to avoid the formation of higher order structures via intermolecular G-tetrads. These INH-ODNs were equally or significantly more efficient to impair TLR7- and TLR9-stimulated murine B-cells, macrophages, conventional and plasmacytoid dendritic cells than the parent INH-ODN 2088, which lacks G-modification. Here, we evaluate the inhibitory/therapeutic potential of our set of G-modified INH-ODN on human immune cells. We report the novel finding that G-modified INH-ODNs efficiently inhibited the release of IFN-α by PBMC stimulated either with the TLR7-ligand oligoribonucleotide (ORN) 22075 or the TLR9-ligand CpG-ODN 2216. G-modification of INH-ODNs significantly improved inhibition of IL-6 release by PBMCs and purified human B-cells stimulated with the TLR7-ligand imiquimod or the TLR9-ligand CpG-ODN 2006. Furthermore, inhibition of B-cell activation analyzed by expression of activation markers and intracellular ATP content was significantly improved by G-modification. As observed with murine B-cells, high concentrations of INH-ODN 2088 but not of G-modified INH-ODNs stimulated IL-6 secretion by PBMCs in the absence of TLR-ligands thus limiting its blocking efficacy. In summary, G-modification of INH-ODNs improved their ability to impair TLR7- and TLR9-mediated signaling in those human immune cells which are considered as crucial in the pathophysiology of SLE.