Proteolytic enzymes in human fetal membranes and amniotic fluid. A comparison of normal and premature ruptured membranes

The amniotic and chorionic membranes obtained at term and term amniotic fluid contain a soluble protease activity which cleaves [14C]-labeled globin at acid pH. In contrast, a salt extract of the pellet fraction obtained from the fetal membranes displays only negligible protease activities at the pH range of 4-8. Specific activities of the proteases in the soluble and salt-extractable fractions of fetal membranes which were intact before onset of labor were not significantly different from the respective activities in cases of premature rupture of fetal membranes (PROM). However, the protease activity of the amniotic fluid was found to increase with advancing gestational age and to reach maximal activity at term. A heat-sensitive and nondializable protease inhibitory activity was found in term amniotic fluid. This inhibitory activity acted on the cytosolic protease of amniotic membranes from control and PROM cases, but not on the soluble protease of chorionic membranes, and had a similar potency in fluids from PROM cases or fluids collected at term. These results do not support a role for fetal membrane proteases, amniotic fluid proteases, or amniotic fluid protease inhibitory activities in the etiology of PROM. However, the observed changes in amniotic fluid protease activity with fetal age suggest a physiological role for the enzyme in normal fetal development.     

HMGB1 Mediates Endogenous TLR2 Activation and Brain Tumor Regression

Background

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs.

Methods and Findings

Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4⁺ and CD8⁺ T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1.

Conclusions

Our data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.     

Syntaxin 4 is required for acid sphingomyelinase activity and apoptotic function

Acid sphingomyelinase (A-SMase) is an important enzyme in sphingolipid metabolism and plays key roles in apoptosis, immunity, development, and cancer. In addition, it mediates cytotoxicity of cisplatin and some other chemotherapeutic drugs. The mechanism of A-SMase activation is still undefined. We now demonstrate that, upon CD95 stimulation, A-SMase is activated through translocation from intracellular compartments to the plasma membrane in an exocytic pathway requiring the t-SNARE protein syntaxin 4. Indeed, down-regulation of syntaxin 4 inhibits A-SMase translocation and activation induced by CD95 stimulation. This leads to inhibition of the CD95-triggered signaling events, including caspase 3 and 9 activation and apoptosis, activation of the survival pathway involving the protein kinase Akt, and important changes in cell cycle and proliferation. The molecular interaction between A-SMase and syntaxin 4 was not known and clarifies the mechanism of A-SMase activation. The novel actions of syntaxin 4 in sphingolipid metabolism and exocytosis we describe here define signaling mechanisms of broad relevance in cell pathophysiology.     

Cutting edge: PHLPP regulates the development, function, and molecular signaling pathways of regulatory T cells

Regulatory T cells (Tregs) have a reduced capacity to activate the PI3K/Akt pathway downstream of the TCR, and the resulting low activity of Akt is necessary for their development and function. The molecular basis for the failure of Tregs to activate Akt efficiently, however, remains unknown. We show that PH-domain leucine-rich-repeat protein phosphatase (PHLPP), which dephosphorylates Akt, is upregulated in Tregs, thus suppressing Akt activation. Tregs expressed higher levels of PHLPP than those of conventional T cells, and knockdown of PHLPP1 restored TCR-mediated activation of Akt in Tregs. Consistent with their high Akt activity, the suppressive capacity of Tregs from PHLPP1(-/-) mice was significantly reduced. Moreover, the development of induced Tregs was impaired in PHLPP1(-/-) mice. The increased level of Akt's negative regulator, PHLPP, provides a novel mechanism used by T cells to control the Akt pathway and the first evidence, to our knowledge, for a molecular mechanism underlying the functionally essential reduction of Akt activity in Tregs.     

Prevalence of T790M mutation among TKI-therapy resistant Lebanese lung cancer patients based on liquid biopsy analysis: a first report from a major tertiary care center

Molecular Biology Reports - Lung adenocarcinoma patients have variable prognosis due to many factors. Detection of epidermal growth factor receptor (EGFR) activating mutations is one of the factors...     

Distribution of spore-positive and spore-negative nodules in stands ofAlnus glutinosa andAlnus incana in Finland

Summary The distribution of spore positive (Sp⁺) and spore negative (Sp⁻) nodules on the two native alder species (A. incana andA. glutinosa) in Finland was investigated. Nodules were collected throughout the country from different ecosystems (forests, swamps, lake- sea- and riversides, old pastures and fields as well as from alder plantations). OnA. incana Sp⁺ nodules predominated, whereas onA. glutinosa the vast majority of the nodules were of the Sp⁻ type. Sp⁺ nodules onA. glutinosa were found only at sites where the two alder species grew close together. This distribution pattern indicates an association of nodule type with alder species, the reasons for which are discussed. Indications of saprophytic growth in the Sp⁻ strain were also found.     

Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.     

Performance Evaluation of Shared Mesh Protection in WDM Networks

The efficient use of network capacity in shared restoration schemes strongly depends upon the path selection procedure. In this paper we propose and evaluate path selection algorithms for sharable and restorable connections in optical networks. Namely, two distributed path selection algorithms are proposed. The first approach maintains global information on network resource usage to determine link sharability and compute optimal shared paths. The second approach only relies upon local information maintained at each node. Subsequently, we present an analytical model to evaluate the performance of these path selection algorithms and show its accuracy through numerical examples. Results indicate that path selection algorithms that maximally exploit the use of reserved sharable channels do not necessarily result in fast restoration; rather these two optimality criteria can conflict most of the time. Additionally, algorithms that maximally exploit the sharability condition typically result in lower scalability and higher complexity.