Cutting edge: Species-specific TLR9-mediated recognition of CpG and non-CpG phosphorothioate-modified ohgonucleotides

Different DNA motifs are required for optimal stimulation Of mouse and human immune cells by CpG oligode-oxynucleotides (ODN). These species differences presumably reflect sequence differences in TLR9, the CPG DNA receptor. In this study, we show that this sequence specificity is restricted to phosphorothioate (PS)-modified ODN and is not observed when a natural phosphodiester backbone is used. Thus, human and mouse cells have not evolved to recognize different CpG motifs in natural DNA. Nonoptimal PS-ODN (i.e., mouse CpG motif on human cells and vice versa) gave delayed and less sustained phosphorylation of p38 AWK than optimal motifs. When the CpG dinucleotide was inverted to GC In each ODN some residual activity of the PS-ODN was retained in a species-specific, TLR-9-dependent manner. Thus, TLR9 may he responsible for mediating many published CpG-independent responses to PS-ODN.

Modelling cell lifespan and proliferation: is likelihood to die or to divide independent of age?

In cell lifespan studies the exponential nature of cell survival curves is often interpreted as showing the rate of death is independent of the age of the cells within the population. Here we present an alternative model where cells that die are replaced and the age and lifespan of the population pool is monitored until a, steady state is reached. In our model newly generated individual cells are given a determined lifespan drawn from a number of known distributions including the lognormal, which is frequently found in nature. For lognormal lifespans the analytic steady-state survival curve obtained can be well-fit by a single or double exponential, depending on the mean and standard deviation. Thus, experimental evidence for exponential lifespans of one and/or two populations cannot be taken as definitive evidence for time and age independence of cell survival. A related model for a dividing population in steady state is also developed. We propose that the common adoption of age-independent, constant rates of change in biological modelling may be responsible for significant errors, both of interpretation and of mathematical deduction. We suggest that additional mathematical and experimental methods must be used to resolve the relationship between time and behavioural changes by cells that are predominantly unsynchronized.

Unicellular pheromone glands of the pentatomid bug Nezara viridula (Heteroptera : Insecta): Ultrastructure, classification, and proposed function

Male Nezara viridula produce sex pheromones from many independent single cells, each with a duct that opens onto the ventral abdominal surface. Despite the presence of along duct and an associated end complex (in the form of a cupule and microvillus saccule), the structural organization of the cells that comprise the gland conform to Class 1 epidermal gland cell classification : a single cell surrounds the entire secretory complex. Each cuticular cupule contains a central bed of filaments and opens into a narrow tubular ductule that leads from the base of the cupule through the epidermis to the cuticle to open externally as a pore. The cuticle of the cupule is continuous with that of the ductule and has the appearance of three layers, although the inner (middle) layer may be a gap formed during construction of the complex. In young adult males, just molted, the ultrastructure of the cells and their inclusions indicate that they are not active. The region of the cell that is distal to the abdominal cuticle is reduced and the proximal region, surrounding the duct, is enlarged when compared with sexually mature (3-4 weeks old) adult males. At maturity the pheromone cells are enlarged distally around the cupule, but are reduced to a narrow sleeve proximally, around the ductule. Two characteristic cell profiles are evident, based on the shape of the cupule and the organelle content. Type A shows a broad opening to the cupule, an abundance of mitochondria, and few vesicular bodies. Type B has an elongated, narrow, vase-like opening to the cupule, few mitochondria, and numerous vesicular bodies. Type B cells are smaller and more abundant than Type A. Distribution within the epidermal layer also differs. It is likely that the different types represent cells producing different secretion profiles. However, the secretions retained by the standard fixation protocol within mature cells of both types look similar and appear to collect as crystalline bodies within the lumen. This may represent a common storage mechanism.

DNA Motifs Suppressing TLR9 Responses

Immune cells respond to bacterial DNA containing unmethylated CpG motifs via Toll-like receptor 9 (TLR9). Given the apparent role of TLR9 in development of systemic lupus erythernatosus (SLE), there is interest in the development of TLR9 inhibitors. TLR9-mediated responses are reported to be inhibited by a confusing variety of different DNA sequences and structures. To aid characterization, we have provisionally categorized TLR9-inhibitory oligodeoxynucleoti des (ODN) into 4 classes, on the basis of sequence and probable mode of action. Class I are short G-rich ODN, which show sequence-specific inhibition of all TLR9 responses, and may be direct competitive inhibitors for DNA binding to TLR9. Class II are telomeric repeat motifs that inhibit STAT signaling, and thus are not specific to TLR9 responses. Because Class II ODN are generally made as 24-base phosphorothioate-modified ODN (PS-ODN), they also fall into Class IV, defined as long PS-ODN, which inhibit TLR9 responses in a sequence-nonspecific manner. Class III includes oligo (dG) that forms a 4-stranded structure and inhibits DNA uptake. The Class I G-rich motifs show the most promise as selective and potent TLR9 inhibitors for therapeutic applications.

Periocular and Orbital Amyloidosis. Clinical Characteristics, Management, and Outcome

Objective: To present the clinical features and management outcome in a large series of patients with periocular and orbital amyloidosis. Design: Retrospective, noncomparative, interventional case series. Patients: All patients diagnosed with periocular and orbital amyloidosis in 6 oculoplastic and orbital units. Methods: Clinical records of all patients were reviewed. Main Outcome Measures: Clinical presentation, radiological and histological findings, treatment modalities, and outcome. Results. The study included 24 patients (15 female, 9 male) with a mean age of 57 17 years. Nineteen cases were unilateral, and 5 were bilateral. Clinical signs and symptoms included a visible or palpable periocular mass or tissue infiltration (95.8%), ptosis (54.2%), periocular discomfort or pain (25%), proptosis or globe displacement (21%), limitations in ocular motility (16.7%), recurrent periocular subcutaneous hemorrhages (12.5%), and diplopia (8.3%). Seven cases had orbital involvement, and 17 were periocular. Immunohistochemistry in 7 patients showed B cells or plasma cells producing monoclonal immunoglobulin chains that were deposited as amyloid light chains. Only 1 patient was diagnosed with systemic amyloid light chain amyloidosis. Treatment modalities were mainly observation and surgical debulking. During a mean follow-up period of 39 months, 21% showed significant progression after treatment, whereas 79% were stable or showed no recurrence after treatment. Conclusion: Periocular and orbital amyloidosis may present with a wide spectrum of clinical findings and result in significant ocular morbidity. Complete surgical excision is not feasible in many cases, and the goal of treatment is to preserve function and to prevent sight-threatening complications.

Innate immune surveillance of spontaneous B cell lymphomas by natural killer cells and gamma delta T cells

Few studies have demonstrated that innate lymphocytes play a major role in preventing spontaneous tumor formation. We evaluated the development of spontaneous tumors in mice lacking beta-2 microglobulin (beta2m; and thus MHC class I, CD1d, and CD16) and/or perform, since these tumor cells would be expected to activate innate effector cells. Approximately half the cohort of perform gene-targeted mice succumbed to spontaneous disseminated B cell lymphomas and in mice that also lacked beta2m, the lymphomas developed earlier (by more than 100 d) and with greater incidence (84%). B cell lymphomas from perforin/beta2m gene-targeted mice effectively primed cell-mediated cytotoxicity and perform, but not IFN-gamma, IL-12, or IL-18, was absolutely essential for tumor rejection. Activated NK1.1(+) and gammadeltaTCR(+) T cells were abundant at the tumor site, and transplanted tumors were strongly rejected by either, or both, of these cell types. Blockade of a number of different known costimulatory pathways failed to prevent tumor rejection. These results reflect a critical role for NK cells and gammadeltaTCP(+) T cells in innate immune surveillance of B cell lymphomas, mediated by as yet undetermined pathway(s) of tumor recognition.

Inhibition of NF kappa B leads to an intracellular reducing environment in human monocyte-derived immature dendritic cells

Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.

A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is detectable in normal human sera

CD83 is an inducible glycoprotein expressed predominantly by dendritic cells (DC) and B lymphocytes. Expression of membrane CD83 (mCD83) is widely used as a marker of differentiated/ activated DC but its function and ligand(s) are presently unknown. We report the existence of a soluble form of CD83 (sCD83). Using both a sCD83-specific ELISA and Western blotting, we could demonstrate the release of sCD83 by mCD83(+) B cell and Hodgkin's disease-derived cell lines, but not mCD83(-) cells. Inhibition of de novo protein synthesis did not affect the release of sCD83 during short-term (2 h) culture of cell lines although mCD83 expression was significantly reduced, suggesting sCD83 is generated by the release of mCD83. Isolated tonsillar B lymphocytes and monocyte-derived DC, which are mCD83(low), released only low levels of sCD83 during culture. However, the differentiation/activation of these populations both up-regulated mCD83 and increased sCD83 release significantly. Analysis of sera from normal donors demonstrated the presence of low levels (121 +/- 3.6 pg/ml) of circulating sCD83. Further studies utilizing purified sCD83 and the analysis of sCD83 levels in disease may provide clues to the function and ligand(s) of CD83.

Epstein-Barr virus-mediated protection against etoposide-induced apoptosis in BJA-B B cell lymphoma cells: role of Bcl-2 and caspase proteins

Epstein-Barr virus (EBV)-infected B cell lymphomas are resistant to apoptosis during cancer development and treatment with therapies. The molecular controls that determine why EBV infection causes apoptosis resistance need further definition. EBV-positive and EBV-negative BJA-B B cell lymphoma cell lines were used to compare the expression of selected apoptosis-regulating Bcl-2 and caspase proteins in EBV-related apoptosis resistance, after 8 hr or 18-24 hr etoposide treatment (80 muM). Apoptosis was quantified using morphology and verified with Hoechst 33258 nuclear stain and electron microscopy. Fluorescence activated cell sorting (FACS) was used to analyse effects on cell cycle of the EBV infection as well as etoposide treatment. Anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bax, caspase-3 and caspase-9 expression and activation were analysed using Western immunoblots and densitometry. EBV-positive cultures had significantly lower levels of apoptosis in untreated and etoposide-treated cultures in comparison with EBV-negative cultures (p < 0.05). FACS analysis indicated a strong G2/M block in both cell sublines after etoposide treatment. Endogenous Bcl-2 was minimal in the EBV-negative cells in comparison with strong expression in EBV-positive cells. These levels did not alter with etoposide treatment. Bcl-XL was expressed endogenously in both cell lines and had reduced expression in EBV-negative cells after etoposide treatment. Bax showed no etoposide-induced alterations in expression. Pro-caspase-9 and -3 were seen in both EBV-positive and -negative cells. Etoposide induced cleavage of caspase-9 in both cell lines, with the EBV-positive cells having proportionally less cleavage product, in agreement with their lower levels of apoptosis. Caspase-3 cleavage occurred in the EBV-negative etoposide-treated cells but not in the EBV-positive cells. The results indicate that apoptosis resistance in EBV-infected B cell lymphomas is promoted by an inactive caspase-3 pathway and elevated expression of Bcl-2 that is not altered by etoposide drug treatment.

T cells signaled by NF-kappa B- dendritic cells are sensitized not anergic to subsequent activation

Paradoxically, while peripheral self-tolerance exists for constitutively presented somatic self Ag, self-peptide recognized in the context of MHC class II has been shown to sensitize T cells for subsequent activation. We have shown that MHC class II(+)CD86(+)CD40(-) DC, which can be generated from bone marrow in the presence of an NF-kappaB inhibitor, and which constitutively populate peripheral tissues and lymphoid organs in naive animals, can induce Ag-specific tolerance. In this study, we show that CD40(-) human monocyte-derived dendritic cells (DC), generated in the presence of an NF-kappaB inhibitor, signal phosphorylation of TCRzeta, but little proliferation or IFN-gamma in vitro. Proliferation is arrested in the G(1)/G(0) phase of the cell cycle. Surprisingly, responding T cells are neither anergic nor regulatory, but are sensitized for subsequent IFN-gamma production. The data indicate that signaling through NF-kappaB determines the capacity of DC to stimulate T cell proliferation. Functionally, NF-kappaB(-)CD40(-)class II+ DC may either tolerize or sensitize T cells. Thus, while CD40(-) DC appear to prime or prepare T cells, the data imply that signals derived from other cells drive the generation either of Ag-specific regulatory or effector cells in vivo.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-B-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.

Nanocomposite Nafion-Silica membranes for direct methanol fuel cells

Commercially available proton exchange membranes such as Nafion do not meet the requirements for high power density direct methanol fuel cells, partly due to their high methanol permeability. The aim of this work is to develop a new class of high-proton conductivity membranes, with thermal and mechanical stability similar to Nafion and reduced methanol permeability. Nanocomposite membranes were produced by the in-situ sol-gel synthesis of silicon dioxide particles in preformed Nafion membranes. Microstructural modification of Nafion membranes with silica nanoparticles was shown in this work to reduce methanol crossover from 7.48x10-6 cm2s^-1 for pure Nafion® to 2.86 x10-6 cm2s^-1 for nanocomposite nafion membranes (Methanol 50% (v/v) solution, 75 degrees C). Best results were achieved with a silica composition of 2.6% (w/w). We propose that silica inhibits the conduction of methanol through Nafion by blocking sites necessary for methanol diffusion through the polymer electrolyte membrane. Effects of surface chemistry, nanoparticle formation and interactions with Nafion matrix are further addressed.

Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells

A method is presented for the direct extraction of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R-3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (

Comparative in vitro effects of closantel and selected beta-ketoamide anthelmintics on a gastrointestinal nematode and vertebrate liver cells

PNU-87407 and PrNU-88509, beta-ketoamide anthelmintics that are structurally related to each other and to the salicylanilide anthelmintic closantel, exhibit different anthelmintic spectra and apparent toxicity in mammals, The basis for this differential pharmacology was examined in experiments that measured motility and adenosine triphosphate (ATP) levels in larval and adult stages of the gastrointestinal nematode, Haemonchus contortus, and in a vertebrate liver cell line and mitochondria, PNU-87407 and PNU-88509 both exhibited functional cross-resistance with closantel in larval migration assays using closantel-resistant and -sensitive isolates of H, contortus. Each compound reduced motility and,ATP levels in cultured adult H. contortus in a concentration- and time-dependent manner: however, motility was reduced more rapidly by PNU-88509, and ATP levels were reduced by lower concentrations of closantel than the beta-ketoamides. Tension recordings from segments of adult H, contortus showed that PNU-88509 induces spastic paralysis, while PNU-87407 and closantel induce flaccid paralysis of the somatic musculature. Marked differences in the actions of these compounds were also observed in the mammalian preparations. In Chang liver cells, ATP levels were reduced after 3 h exposures to greater than or equal to 0.25 mu M PNU-87407 1 mu M closantel or 10 mu M PNU-88509, Reductions in ATP caused by PNU-88509 were completely reversible, while the effects of closantel and PNU-87407; were irreversible. PNU-87407, closantel and PNU-88509 uncoupled oxidative phosphorylation in isolated rat liver mitochondria, inhibiting the respiratory control index (with glutamate or succinate as substrate) by 50% at concentrations of 0.14, 0.9 and 7.6 mu M respectively.