Tau assembly in inducible transfectants expressing wild-type or FTDP-17 tau

Conditional expression systems for 4-repeat wild-type (WT) tau or the corresponding mutants V337M and R406W were established in human neuroglioma H4 cells to study the effect of tau mutations on the physicochemical properties of tau, and to develop a cellular model for the formation of filamentous tau characteristic of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease. Upon induction tau expression increased, reaching maximal levels at 5 to 7 days. WT tau was phosphorylated at amino acids T181, S202/T205, T231, and S396/S404. The R406W mutation decreased tau phosphorylation at each of these sites as did the V337M mutation except for S396/S404 sites that increased. Most tau in postnuclear cell lysates was recovered in the supernatant fraction after centrifugation at 200,000 x g. The amount of tau in the pellet fraction increased more in mutant transfectants compared to WT when the induction was extended beyond 5 days. This particulate tau could be partially extracted with salt, Triton X-100, or sarkosyl. Of the transfectants, R406W had the highest proportion of sarkosyl-insoluble tau by day 7. This insoluble fraction was thioflavin S-positive and contained 15- to 5-nm-wide filaments with tau immunoreactivities. The R406W filaments were more abundant than those detected in similar preparations from WT or V337M transfectants. At the light microscopy level, most tau was found with microtubules, or diffusely distributed in the cytoplasm, but none of this appeared thioflavin S-positive. The results suggest that conditional tau transfectants are in a pretangle stage making them an attractive model system for studying intracellular tangle accumulation and for testing potential therapeutic agents as inhibitors for tau aggregation.     

Continuous Glucose Monitoring Use in Clinical Trials for On-Market Diabetes Drugs

To the best of our knowledge, there are no published data on the historical and recent use of CGM in clinical trials of pharmacological agents used in the treatment of diabetes. We analyzed 2,032 clinical trials of 40 antihyperglycemic therapies currently on the market with a study start date between 1 January 2000 and 31 December 2019. According to ClinicalTrials.gov, 119 (5.9%) of these trials used CGM. CGM usage in clinical trials has increased over time, rising from <5% before 2005 to 12.5% in 2019. However, it is still low given its inclusion in the American Diabetes Association’s latest guidelines and known limitations of A1C for assessing ongoing diabetes care.

The availability of reliable continuous glucose monitoring (CGM) systems has proven to be a major innovation in diabetes management and research. Most current CGM systems are approved for 7- to 14-day use and use a wire-tipped glucose oxidase sensor inserted in subcutaneous tissue to monitor glucose concentrations in interstitial fluid. One implanted CGM system is approved for longer-term use (90–180 days); it operates with fluorescence-based technology. CGM sensors record a glucose data point every 1–15 minutes (depending on the system), collecting far more granular data and information on glycemic patterns than self-monitoring of blood glucose (SMBG) alone. Real-time CGM or intermittently scanned CGM systems send data continuously or intermittently to dedicated receivers or smartphones, whereas professional CGM systems provide retrospective data, either blinded or unblinded, for analysis and can be used to identify patterns of hypo- and hyperglycemia. Professional CGM can be helpful to evaluate patients when other CGM systems are not available to the patient or the patient prefers a blinded analysis or a shorter experience with unblinded data.In the 20 years since CGM systems first became available to people with diabetes, technological improvements, particularly pertaining to accuracy and form factor, have made CGM increasingly viable for both patient use and clinical investigation (1,2). Average sensor MARD (mean absolute relative difference; a summary accuracy statistic) has decreased from >20 to <10% (3–10), including two systems that do not require fingerstick calibrations and three that are approved to be used for insulin dosing (11). Concurrently, size, weight, and cost of CGM systems have all decreased, while user-friendliness and convenience have increased (12).To encourage use of CGM-derived data, researchers and clinicians have worked to develop a standard set of glycemic metrics beyond A1C. In 2017, two international groups of leading diabetes clinical and research organizations published consensus definitions for key metrics, including clinically relevant glycemic cut points for hypoglycemia (<70 and <54 mg/dL), hyperglycemia (>180 and >250 mg/dL), and time in range (TIR; 70–180 mg/dL) (13,14).CGM-derived metrics provide far greater precision and granularity than is possible with SMBG or A1C data alone (Table 1), enabling clinicians and investigators to better represent inter- and intraday glycemic differences with metrics such as TIR, glycemic variability, and time in hypoglycemia and hyperglycemia (15). Crucially, CGM also allows for the accurate measurement and detection of nocturnal glycemia (16). The use of these metrics enables a more comprehensive understanding of glycemic management that can facilitate individualized treatment for people with diabetes or prediabetes. Although A1C is a useful estimate of mean glucose over the previous 2–3 months, especially when evaluating population health, it is important to include other glycemic outcomes in clinical trials. Furthermore, there is emerging evidence suggesting that TIR predicts the development of microvascular complications at least as well as A1C (17,18).TABLE 1Benefits of CGM Compared With A1C Alone in Assessing Glycemia

Increased central adiposity is associated with pro-inflammatory immunoglobulin G N-glycans

Background

Increased body fat may be associated with an increased risk of developing an underlying pro-inflammatory state, thus leading to greater risk of developing certain chronic conditions. Immunoglobulin G has the ability to exert both anti- and pro-inflammatory effects, and the N-glycosylation of the fragment crystallisable portion is involved in mediating this process. Body mass index, a rudimentary yet gold standard indication for body fat, has been shown to be associated with agalactosylated immunoglobulin G N-glycans.

Function-blocking antibodies to human vascular adhesion protein-1: a potential anti-inflammatory therapy

Human vascular adhesion protein-1 (VAP-1) is a homodimeric 170-kDa sialoglycoprotein that is expressed on the surface of endothelial cells and functions as a semicarbazide-sensitive amine oxidase and as an adhesion molecule. Blockade of VAP-1 has been shown to reduce leukocyte adhesion and transmigration in in vivo and in vitro models, suggesting that VAP-1 is a potential target for anti-inflammatory therapy. In this study we have constructed mouse-human chimeric antibodies by genetic engineering in order to circumvent the potential problems involved in using murine antibodies in man. Our chimeric anti-VAP-1 antibodies, which were designed to lack Fc-dependent effector functions, bound specifically to cell surface-expressed recombinant human VAP-1 and recognized VAP-1 in different cell types in tonsil. Furthermore, the chimeric antibodies prevented leukocyte adhesion and transmigration in vitro and in vivo. Hence, these chimeric antibodies have the potential to be used as a new anti-inflammatory therapy.     

A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer

BACKGROUND: There is a continued need to develop more effective cancer immunotherapy strategies. Exosomes, cell-derived lipid vesicles that express high levels of a narrow spectrum of cell proteins represent a novel platform for delivering high levels of antigen in conjunction with costimulatory molecules. We performed this study to test the safety, feasibility and efficacy of autologous dendritic cell (DC)-derived exosomes (DEX) loaded with the MAGE tumor antigens in patients with non-small cell lung cancer (NSCLC). METHODS: This Phase I study enrolled HLA A2+ patients with pre-treated Stage IIIb (N = 4) and IV (N = 9) NSCLC with tumor expression of MAGE-A3 or A4. Patients underwent leukapheresis to generate DC from which DEX were produced and loaded with MAGE-A3, -A4, -A10, and MAGE-3DPO4 peptides. Patients received 4 doses of DEX at weekly intervals. RESULTS: Thirteen patients were enrolled and 9 completed therapy. Three formulations of DEX were evaluated; all were well tolerated with only grade 1-2 adverse events related to the use of DEX (injection site reactions (N = 8), flu like illness (N = 1), and peripheral arm pain (N = 1)). The time from the first dose of DEX until disease progression was 30 to 429+ days. Three patients had disease progression before the first DEX dose. Survival of patients after the first DEX dose was 52-665+ days. DTH reactivity against MAGE peptides was detected in 3/9 patients. Immune responses were detected in patients as follows: MAGE-specific T cell responses in 1/3, increased NK lytic activity in 2/4. CONCLUSION: Production of the DEX vaccine was feasible and DEX therapy was well tolerated in patients with advanced NSCLC. Some patients experienced long term stability of disease and activation of immune effectors.     

Dopamine in the orbitofrontal cortex regulates operant responding under a progressive ratio of reinforcement in rats

Prefrontocortical dopamine (DA) plays an essential role in the regulation of cognitive functions and behavior. The orbitofrontal cortex (OFC) receives a dopaminergic projection from the ventral tegmental area and is particularly important for goal-directed appetitive behaviors and for the neural representation of reward value. We here examined the effects of DA receptor blockers locally infused into the OFC, on instrumental behavior under a progressive schedule of reinforcement. After continuous reinforcement training (lever pressing for casein pellets) rats received bilateral intra-OFC-infusions of the DA D1-receptor antagonist SCH23390 (3 μg/0.5 μl), the DA D2-receptor antagonist sulpiride (3 μg/0.5 μl), or phosphate buffered saline through chronically indwelling cannulae. Immediately after infusion they were tested under a time-constrained progressive ratio schedule of reinforcement (3, 6, 9, 12, … lever presses for 1 casein pellet within 180 s). Both SCH23390 and sulpiride led to a significant reduction of the break point (cessation to respond to the increasing criterion of instrumental effort) compared to vehicle infusions. A food preference test revealed no drug effects on the amount of consumed pellets and on the preference of casein pellets over laboratory chow. Leftward shifts of the break point in progressive ratio tasks indicate a disturbance of the mechanisms that translate motivation into appetitive behavior under conditions of increasing instrumental effort. Therefore, our data indicate that orbitofrontal dopamine is necessary for reward-related instrumental behavior.     

Three-dimensional in vitro model of adipogenesis: comparison of culture conditions

In vivo and in vitro studies have demonstrated both promise and current limitations in tissue engineering of fat. Herein, we report the establishment of a well-defined three-dimensional (3-D) in vitro model useful for systematic investigations of 3-D adipogenesis. Polyglycolic acid fiber meshes were dynamically seeded with 3T3-L1 preadipocytes; subsequently, cell-polymer constructs were hormonally induced and cultivation under three different conditions was evaluated. Regarding tissue coherence and intracellular lipid content, culture of cell-polymer constructs either dynamically in well plates or in stirred bioreactors yielded similar results, which were distinctly improved compared with static conditions in well plates. At the protein and mRNA levels, significantly increased expression of genes characteristic for a mature adipose phenotype was demonstrated for constructs dynamically cultured in well plates, as compared with static conditions. Furthermore, investigation of lipolysis under stimulating and inhibiting conditions demonstrated functionality of the dynamically differentiated constructs. Using dynamic culture conditions, the presented in vitro model system is suggested as a valuable tool serving both fat tissue engineering and basic research by facilitating investigations of tissue-inherent features not possible under conventional 2-D culture conditions.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.