Single step determination of PCB 126 and 153 in rat tissues by using solid phase microextraction/gas chromatography–mass spectrometry: Comparison with solid phase extraction and liquid/liquid extraction

A simple and reliable solid phase microextraction/gas chromatography–mass spectrometry (SPME/GC–MS) method was developed for the single-step determination of PCBs 126 and 153 in rat brain and serum, using liquid/liquid and solid phase extraction (SPE) as reference techniques. The multi-factor categorical experimental design used to study simultaneously the main parameters and their interactions affecting the efficiency of the method, showed that the use of an 85 μm PA exposed at 100 °C for 40 min was the optimum sampling condition for both PCBs. SPME was then validated by studying its linear dynamic (over two orders of magnitude), limits of detection (brain: 2 ng/g, serum: 0.2 ng/g) and analytical precision that was within 9% for SPME in both brain and serum. Finally, the method was used to determine the brain and blood target dose in mothers and pups after oral exposure of the mothers.     

PI3Kδ is essential for tumor clearance mediated by cytotoxic T lymphocytes

Background

PI3Kδ is a lipid kinase of the phosphoinositide 3-kinase class 1A family and involved in early signaling events of leukocytes regulating proliferation, differentiation and survival. Currently, several inhibitors of PI3Kδ are under investigation for the treatment of hematopoietic malignancies. In contrast to the beneficial effect of inhibiting PI3Kδ in tumor cells, several studies reported the requirement of PI3Kδ for the function of immune cells, such as natural killer and T helper cells. Cytotoxic T lymphocytes (CTLs) are essential for tumor surveillance. The scope of this study is to clarify the potential impact of PI3Kδ inhibition on the function of CTLs with emphasis on tumor surveillance.

Principal Findings

PI3Kδ-deficient mice develop significantly bigger tumors when challenged with MC38 colon adenocarcinoma cells. This defect is accounted for by the fact that PI3Kδ controls the secretory perforin-granzyme pathway as well as the death-receptor pathway of CTL-mediated cytotoxicity, leading to severely diminished cytotoxicity against target cells in vitro and in vivo in the absence of PI3Kδ expression. PI3Kδ-deficient CTLs express low mRNA levels of important components of the cytotoxic machinery, e.g. prf1, grzmA, grzmB, fasl and trail. Accordingly, PI3Kδ-deficient tumor-infiltrating CTLs display a phenotype reminiscent of naïve T cells (CD69^lowCD62L^high). In addition, electrophysiological capacitance measurements confirmed a fundamental degranulation defect of PI3Kδ−/− CTLs.

Conclusion

Our results demonstrate that CTL-mediated tumor surveillance is severely impaired in the absence of PI3Kδ and predict that impaired immunosurveillance may limit the effectiveness of PI3Kδ inhibitors in long-term treatment.     

Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol

Background aimsWe have recently shown that thymoglobulin (TG) efficiently expands cytokine-induced killer (CIK) cells in combination with interferon (IFN)-γ and interleukin (IL)-2 (ITG2 protocol). It is presently unknown whether the infusion of autologous immune effector cells generated by TG, IFN-γ and IL-2 is feasible and safeMethodsFive patients with advanced and/or refractory solid tumors were enrolled in the present phase I/II study. Peripheral blood mononuclear cells (PBMC) collected by leukapheresis were stimulated under good manufacturing practice (GMP)-conditions with IFN-γ, followed by TG and IL-2. After 2–3 weeks in culture, a median of 4.65 × 10⁶ immune effector cells per kilogram of recipient's body weight was obtained and infused intravenously. The median time from enrollment into the study to infusion of the expanded CIK cells was 30 daysResultsITG2 efficiently expanded immune effector cells that comprised both conventional natural killer (NK) cells and CD3⁺ CD16⁺ CD56⁺ CIK cells. One patient with advanced melanoma died because of disease progression before the infusion of CIK cells. The target dose of at least 2.5 × 10⁶ CIK cells/kg of recipient's body weight was reached in four out of five evaluable patients. CIK cells were administered intravenously without any measurable toxicity. In vitro, CIK cells exerted lytic activity against cervical cancer cells. The median survival was 4.5 months (range 1–13) from the first infusion of CIK cells.ConclusionsThis study has highlighted the feasibility and safety of the administration of CIK cells generated with the ITG2 protocol. Whether CIK cells can help control disease burden in patients with advanced malignancies will be determined in future clinical trials.     

The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS

Remyelination of primary demyelinated lesions is a common feature of experimental models of multiple sclerosis (MS) and is also suggested to be the normal response to demyelination during the early stages of MS itself. Many lines of evidence have shown that remyelination is preceded by the division of endogenous oligodendrocyte precursor cells (OPCs) in the lesion and its borders. It is suggested that this rapid response of OPCs to repopulate the lesion site and their subsequent differentiation into new oligodendrocytes is the key to the rapid remyelination. Antibodies to the NG2 chondroitin sulphate proteoglycan have proved exceedingly useful in following and quantitating the response of endogenous OPCs to demyelination. Here we review the literature on the response of NG2-expressing OPCs to demyelination and provide some new evidence on their response to the chronic inflammatory demyelinating environment seen in recombinant myelin oligodendrocyte glycoprotein (MOG) induced experimental allergic encephalomyelitis (EAE) in the DA rat. NG2-expressing OPCs responded to the inflammatory demyelination in this model by becoming reactive and increasing in number in a very focal manner. Evidence of NG2⁺OPCs in lesioned areas beginning to express the oligodendrocyte marker CNP was also seen. The response of OPCs appeared to occur following successive relapses but did not always lead to remyelination, with areas of chronic demyelination observed in the spinal cord. The presence of OPCs in the adult human CNS is clearly of vital importance for repair in multiple sclerosis (MS). As in rat tissue, the antibody labels an evenly distributed cell population present in both white and grey matter, distinct from HLA-DR⁺microglia. NG2⁺cells are sparsely distributed in the centre of chronic MS lesions. These cells apparently survive demyelination and exhibit a multi-processed or bipolar morphology in the very hypocellular environment of the lesion.     

Laterobasal membranes from intestinal epithelial cells: Isolation free of intracellular membrane contaminants

Summary A simplified method for isolating highly purified laterobasal membranes (LBM) from enterocytes is based on treatment of membranes with 8mm CaCl₂ concentration in order to aggregate intracellular membrane contaminants. The resultant LBM showed an average 15-fold enrichment and constituted 8% of the original K-stimulated phosphatase in the initial crude homogenate. It showed typical LBM migration on counter-current distribution (CCD) and was essentially free of contamination with endoplasmic reticulum and Golgi membranes. This method is highly efficient and yields sufficient purified LBM to allow comprehensive analysis of enterocyte membrane events.     

Marine glycosyl hydrolases in the hydrolysis and synthesis of oligosaccharides

The marine ecosystem can be considered a rather unexplored source of biological material (e.g. natural substances with therapeutic activity) and can also be a surprising source of enzymes carrying new and interesting catalytic activities to be applied in biocatalysis. The use of glycosyl hydrolases from marine environments dates back to the end of the 1960s and was mainly focused on the development of sensitive and reliable hydrolytic methods for the analysis of sugar chains. As a result not all the benefits of a particular enzymatic activity have been investigated, especially regarding the transglycosylation potential of these enzymes for the synthesis of glycosidic bonds. In this review, the potential of marine sources will be demonstrated reporting on the few examples found in literature for the synthesis and hydrolysis of biologically relevant oligosaccharides catalyzed by glycosyl hydrolases of marine origin. Particular emphasis is given to the synthesis of glycosidic bonds, which is easy by the use of glycosyl hydrolases. Further aspects considered in this review are applications of these biocatalysts for vegetal waste treatment in recovering useful materials, for structural identification and for preparation of target materials from new purified polysaccharides, for the synthesis or modification of food-related compounds and for glycobiology related studies.     

Diffuse cutaneous candidiasis in a dog. Diagnosis by PCR-REA

The authors describe a clinical case of cutaneous candidiasis in a dog with dermatological lesions, characterized by persistent alopecia, crusts, ulcers and scales. Predisposing factors such as the use of corticosteroids, the concomitan presence of an autoimmune disease (pemphigus foliaceus) and an infection of ehrlichiosis caused by Ehrlichia canis were observed. Histopathological findings included signs of orthokeratotic hyperkeratosis, moderate follicular keratosis and light epidermic acanthosis. The reactive process included an infiltrative superficial dermatitis and a mural folliculitis with prevalent participation of macrophages and lymphocytes. The application of PCR-Restriction Enzyme Analysis (REA) method on cutaneous specimens in veterinary medicine is an extremely interesting diagnostic tool. Its use, together with other techniques, such as mycologic, cytologic and histological examinations, allowed us to identify Candida albicans as aetiological agent in this particular case.     

DRD2/CHRNA5 Interaction on Prefrontal Biology and Physiology during Working Memory

Background

Prefrontal behavior and activity in humans are heritable. Studies in animals demonstrate an interaction between dopamine D2 receptors and nicotinic acetylcholine receptors on prefrontal behavior but evidence in humans is weak. Therefore, we hypothesize that genetic variation regulating dopamine D2 and nicotinic acetylcholine receptor signaling impact prefrontal cortex activity and related cognition. To test this hypothesis in humans, we explored the interaction between functional genetic variants in the D2 receptor gene (DRD2, rs1076560) and in the nicotinic receptor α5 gene (CHRNA5, rs16969968) on both dorsolateral prefrontal cortex mediated behavior and physiology during working memory and on prefrontal gray matter volume.

Methods

A large sample of healthy subjects was compared for genotypic differences for DRD2 rs1076560 (G>T) and CHNRA5 rs16969968 (G>A) on prefrontal phenotypes, including cognitive performance at the N-Back task, prefrontal physiology with BOLD fMRI during performance of the 2-Back working memory task, and prefrontal morphometry with structural MRI.

Results

We found that DRD2 rs1076560 and CHNRA5 rs16969968 interact to modulate cognitive function, prefrontal physiology during working memory, and prefrontal gray matter volume. More specifically, CHRNA5-AA/DRD2-GT subjects had greater behavioral performance, more efficient prefrontal cortex activity at 2Back working memory task, and greater prefrontal gray matter volume than the other genotype groups.

Conclusions

The present data extend previous studies in animals and enhance our understanding of dopamine and acetylcholine signaling in the human prefrontal cortex, demonstrating interactions elicited by working memory that are modulated by genetic variants in DRD2 and CHRNA5.