Culture Cell Lysates Research Articles

Evidence for the extracellular delivery of influenza NS1 protein

We constructed a reporter influenza A/Puerto Rico/8/1934 virus expressing truncated 124aa N-terminal NS1 protein fused to a luciferase reporter sequence (NanoLuc) without signal peptide. The reproduction activity of the vector correlated well with the luminescent activity in the lysates of infected cell cultures or mouse respiratory organ suspensions. Surprisingly, we found that luciferase enzymatic activity was present not only in the intracellular compartments but also in cell culture supernatants as well as in the sera or bronchiolar lavages of infected mice. This fact allowed us to formulate a working hypothesis about the extracellular delivery mechanism of the NS1 protein. To test this idea, we conducted co-transfection experiments in Vero cells with different combinations of plasmids encoding influenza genomic segments and chimeric NS1-NanoLuc encoding plasmid. We found that the emergence of the luciferase reporter in the extracellular compartment was promoted by the formation of the ribonucleoprotein complex (RNP) from the co-transfection of plasmids expressing PB1, PB2, PA, and NP proteins. Therefore, influenza NS1 protein may be delivered to the extracellular compartment together with the nascent RNP complexes during the maturation of virus particles.

A novel approach for the development of bio-sensitized solar cell using cell lysate of a haloarchaeon Halostagnicola larsenii RG2.14 (MCC 2809) containing bacteriorhodopsin

Bacteriorhodopsin (BR) present in the cell membrane of haloarchaea and capable of converting light energy into electrical energy is being explored as biosensitizer in bio-sensitized solar cells (BSSC). The haloarchaeon, Halostagnicola larsenii RG2.14 (MCC 2809) containing bacteriorhodopsin (BR), was used to prepare BSSC. The cell lysate of the culture containing BR was immobilized on TiO2 nanocrystalline films. The BSSC was characterized for photoelectric performance, UV–visible spectroscopy, Scanning electron microscopy, and chronopotentiometry-chronoamperometry. Studies on the parameters like the use of ethanol for the cell lysate preparation resulted in the maximum open-circuit voltage of 0.52 V, short circuit photocurrent density of 120 μA/cm2 fill factor of 0.54 and overall efficiency of 0.11%. The present investigation indicates that the BR containing haloarchaeal culture cell lysate could be used in the preparation of BSSC.

Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin).

Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. We performed western blotting for transmembrane iron transporter proteins; SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.

Trehalose improves PPR vaccine virus stability in diluent

Background: Specialized freeze-drying process is being used in the field for different thermostable vaccine preparation worldwide. The thermostability remains only in undiluted conditions. If dilution is made at the morning and used for the whole day, the vaccine efficacy is compromised at high ambient temperature. In this study, trehalose based specialized vaccine diluent was used to improve the stability of Peste des Petits Ruminants (PPR) vaccine in diluted condition. Methods: The available PPR vaccine was reconstituted with conventional diluent and with trehalose based test diluent. The diluted vaccine was kept at ambient temperature without maintaining any cool chain. Stability of diluted vaccine virus was further assessed in vivo and in vitro at different temperatures. Goats were vaccinated and Vero cells were infected with reconstituted vaccines and were assessed at 0, 3, 6, 9 and 24 hours post dilution. Antibody titer was measured and virus infectivity titer was determined in both cultured cell lysate and supernatant. The presence of the virus particles in Vero cell was confirmed by standard RT-PCR targeting Fusion (F) gene of PPR virus. Results: In vivo results revealed that the number of goats possessed antibodies to PPR virus was higher in trehalose based vaccine formulation than the conventional PBS based diluent. Reconstituted vaccine virus (using PBS and trehalose diluent) infected Vero cells produced 70-80% cytopathic effect (CPE) in 5th days of post infection. Both diluents produced and maintained infectivity titer from log10 TCID50 5.5 to log10 TCID50 3.6, until the use of vaccines incubated for 9 hours after dilution. On the other hand, at 24 hours of post dilution only trehalose formulated vaccine produced log10 TCID502.5 whereas no infectivity titer was observed at the same time using conventional one. Conclusion: The present study suggests that trehalose preserves the quality of reconstituted vaccine in terms of infectivity titers. Trehalose can be a diluent of choice for reconstitution of PPR vaccine in field.

SAT-558 Tsh Modulation Of Bone Biology - Further Evidence From A Recombinant Tsh-β Variant.

We have previously shown that TSH has modulatory influences on osteoblasts and osteoclasts and can act as a bone protection ligand. We have also shown that a novel TSH-β variant protein is produced locally by murine and human bone marrow macrophages (Endocrinology 154:4919-26, 2013 and Endocrinology 157:3658-67, 2016). Furthermore, we have also shown by molecular modelling that both TSH-β and TSH-βv can bind to the native TSH receptor. In order to characterize the biological activity of this TSH-β variant we have inserted the gene sequence together with a FLAG tag into the P-secTag2 vector which was then cloned into mammalian HEK-293 cells. Western blot analysis identified 17KD and a 12KD proteins corresponding to the wild type TSH-β and the TSH-βv nucleotide sequence in cell culture lysates and, less so in supernatants, by using a FLAG monoclonal antibody and a TSH-β polyclonal antibody. Protein bands of ~34 KD and 24 KD were observed only in the absence of DTT suggesting dimeric forms of TSH-β and TSH-βv respectively. Detection by Coomassie staining followed by sequence analysis using mass spectrometry confirmed the identity of these proteins after column purification. Furthermore, cyclic AMP responses were generated by both supernatant preparations with an 8-fold increase in luciferase activity with TSH-β and a 4-fold increase with TSH-βv. These data demonstrate the inherent bioactivity of single TSH-β chains which was mirrored by TSH-βv. Local paracrine action by macrophage derived TSH-βv on osteoblasts and osteoclasts is, therefore, most likely and raises the possibility of this variant form having significant osteoprotective activity.

The use of in vitro transcriptional data to identify thresholds of effects in a human lens epithelial cell-line exposed to ionizing radiation

Purpose: The International Commission on Radiological Protection (ICRP) recently recommended reducing the occupational equivalent dose limit for the lens of the eye. Based primarily on a review of epidemiological data, the absorbed dose threshold is now considered to be 0.5 Gy independent of dose-rate and severity of opacification, reduced from the previous threshold of 2 Gy. However, direct mechanistic evidence to support an understanding of the underlying molecular mechanisms of damage is still lacking. To this end, we explored the effects of a broad dose-range of ionizing radiation exposure on gene expression changes in a human lens epithelial (HLE) cell-line in order to better understand the shape of the dose–response relationship and identify transcriptional thresholds of effects.Methods: HLE cells were exposed to doses of 0, 0.01, 0.05, 0.25, 0.5, 2, and 5 Gy of X-ray radiation at two dose rates (1.62 cGy/min and 38.2 cGy/min). Cell culture lysates were collected 20 h post-exposure and analyzed using whole-genome RNA-sequencing. Pathways and dose-thresholds of biological effects were identified using benchmark dose (BMD) modeling.Results: Transcriptional responses were minimal at doses less than 2 Gy. At higher doses, there were a significant number of differentially expressed genes (DEGs) (p≤.05, fold change≥|1.5|) at both dose rates, with 1308 DEGs for the low dose rate (LDR) and 840 DEGs for the high dose rate (HDR) exposure. Dose–response modeling showed that a number of genes exhibited non-linear bi-phasic responses, which was verified by digital droplet PCR. BMD analysis showed the majority of the pathways responded at BMD median values in the dose range of 1.5–2.5 Gy, with the lowest BMD median value being 0.6 Gy for the HDR exposure. The minimum pathway BMD median value for LDR exposure, however, was 2.5 Gy. Although the LDR and HDR exposures shared pathways involved in extracellular matrix reorganization and collagen production with BMD median value of 2.9 Gy, HDR exposures were more effective in activating pathways associated with DNA damage response, apoptosis, and cell cycling relative to LDR exposure.Conclusions: Overall, the results suggest that radiation induces complex non-linear transcriptional dose–response relationships that are dose-rate dependent. Pathways shared between the two dose rates may be important contributors to radiation-induced cataractogenesis. BMD analysis suggests that the majority of pathways are activated above 0.6 Gy, which supports current ICRP identified dose thresholds for deterministic effects to the lens of the eye of 0.5 Gy.

Angiopoietin-like 4 promotes the intracellular cleavage of lipoprotein lipase by PCSK3/furin in adipocytes

Lipoprotein lipase (LPL) catalyzes the breakdown of circulating triglycerides in muscle and fat. LPL is inhibited by several proteins, including angiopoietin-like 4 (ANGPTL4), and may be cleaved by members of the proprotein convertase subtilisin/kexin (PCSK) family. Here, we aimed to investigate the cleavage of LPL in adipocytes by PCSKs and study the potential involvement of ANGPTL4. A substantial portion of LPL in mouse and human adipose tissue was cleaved into N- and C-terminal fragments. Treatment of different adipocytes with the PCSK inhibitor decanoyl-RVKR-chloromethyl ketone markedly decreased LPL cleavage, indicating that LPL is cleaved by PCSKs. Silencing of Pcsk3/furin significantly decreased LPL cleavage in cell culture medium and lysates of 3T3-L1 adipocytes. Remarkably, PCSK-mediated cleavage of LPL in adipocytes was diminished by Angptl4 silencing and was decreased in adipocytes and adipose tissue of Angptl4-/- mice. Differences in LPL cleavage between Angptl4-/- and WT mice were abrogated by treatment with decanoyl-RVKR-chloromethyl ketone. Induction of ANGPTL4 in adipose tissue during fasting enhanced PCSK-mediated LPL cleavage, concurrent with decreased LPL activity, in WT but not Angptl4-/- mice. In adipocytes, after removal of cell surface LPL by heparin, levels of N-terminal LPL were still markedly higher in WT compared with Angptl4-/- adipocytes, suggesting that stimulation of PCSK-mediated LPL cleavage by ANGPTL4 occurs intracellularly. Finally, treating adipocytes with insulin increased full-length LPL and decreased N-terminal LPL in an ANGPTL4-dependent manner. In conclusion, ANGPTL4 promotes PCSK-mediated intracellular cleavage of LPL in adipocytes, likely contributing to regulation of LPL in adipose tissue. Our data provide further support for an intracellular action of ANGPTL4 in adipocytes.

Expression of a 100kDa recombinant protein from Histoplasma capsulatum in the methylotrophic yeast Pichia pastoris

Background: Histoplasmosis is a systemic and endemic mycosis widely distributed in the Americas caused by the dimorphic fungus Histoplasma capsulatum. The infection is usually asymptomatic in immunocompetent individuals. However, immunocompromised patients may contract the disseminated form of the disease, which has a bad prognosis and requires rapid diagnosis and treatment. The definitive diagnosis involves the isolation of H. capsulatum by culture from clinical specimens, which may take up to 4 weeks. In addition, molecular methods are expensive and have low sensitivity and immunoassays present many false-positive results. Thus, the aim of this work is to express Hc100, a specific protein of 100kDa from H. capsulatum, to develop a novel direct immunoassay and to perform characterization studies of this protein as a first approach for the potential development of novel therapeutic strategies. Methods & Materials: The gene that codes for Hc100 was constructed with a secretory signal and a polyhistidine-tag and was expressed in Pichia pastoris X-33 strain. Cell culture supernatants and lysates from different induction times were analyzed by SDS-PAGE, Westernblot and mass spectrometry. Results: A band of the expected size was observed in the supernatants at 24, 48 and 72 h of methanol induction in Coomasie blue stained gels and its identity was confirmed by Westernblot using anti-histidine antibodies and by mass spectrometry. The highest expression level was observed at 24 h of induction. Also, a lower molecular weight band was observed at 48 and 72 h of induction, probably due to degradation processes. Conclusion: P. pastoris proved to be a valid biotechnological tool for the expression of this specific protein, thus encouragingthe national production of novel fungal antigens for the potential development of new rapid diagnostic tests for this clinical relevant form of the histoplasmosis disease.

Human adipose tissue protein analyses using capillary western blot technology

A capillary western blot (Wes®) technology has recently been validated for analyses of cell culture lysate proteins, but whether it is reliable for human tissue proteins is unknown. We compared traditional western blotting to the Wes® capillary western method to quantitate the relative amount of human adipose tissue CD36, the ratio of phosphorylated Erk1/2 (pErk1/2) to total Erk1/2 during insulin clamp or after niacin treatment and the fold increase in pAktS473 (Akt phosphorylation on Ser473) in response to feeding. The results from these two methods were highly correlated (r = 0.932 for CD36, r = 0.905 for pErk1/2:Erk1/2, r = 0.923 for the change in pAkt/Akt, P < 0.001). On Wes® we observed the distinct peaks around the expected molecular weights for these proteins with decreasing peak areas with serial dilutions of loading protein amount. Wes® and traditional western blot both had linear dynamic ranges for CD36, Erk1/2 and Akt. Due to differences in signal responsiveness for pAkt/Akt, we employed a calibrator sample and log transformation of data to allow proper comparisons. The Wes® approach required less sample than the traditional western blot and less technician/assay time, while achieving high sensitivity and good reproducibility. Capillary western technology (Wes®) provides a satisfactory alternative for analyses of human adipose tissue proteins.

Direct Quantification of Cytochromes P450 and Drug Transporters—A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates

The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.

Rapid and versatile Ms-based immunoassays for targeted cytochrome p450 and transporter profiling in tissue and cell culture lysates

Rapid and versatile Ms-based immunoassays for targeted cytochrome p450 and transporter profiling in tissue and cell culture lysates

Human adipose tissue protein analyses using capillary western blot technology

Background/Objectives:A capillary western blot (Wes) technology has recently been validated for analyses of cell culture lysate proteins, but whether it is reliable for human tissue proteins is unknown.Subjects:We compared traditional western blotting to the Wes capillary Western method to quantitate the relative amount of human adipose tissue CD36, the ratio of phosphorylated Erk1/2 (pErk1/2) to total Erk1/2 during insulin clamp or after niacin treatment and the fold increase in pAktS473 (Akt phosphorylation on Ser473) in response to feeding.Results:The results from these two methods were highly correlated (r=0.932 for CD36, r=0.905 for pErk1/2:Erk1/2, r=0.923 for the change in pAkt/Akt, P<0.001). On Wes we observed the distinct peaks around the expected molecular weights for these proteins with decreasing peak areas with serial dilutions of loading protein amount. Wes and traditional western blot both had linear dynamic ranges for CD36, Erk1/2 and Akt. Due to differences in signal responsiveness for pAkt/Akt, we employed a calibrator sample and log transformation of data to allow proper comparisons. The Wes approach required less sample than the traditional western blot and less technician/assay time, while achieving high sensitivity and good reproducibility.Conclusions:Capillary Western technology (Wes) provides a satisfactory alternative for analyses of human adipose tissue proteins.

Effects of electron beam irradiation on murine norovirus-1 in abalone (Haliotis discus hannai) meat and viscera

Many shellfish-borne viral outbreaks have been reported in East Asia, where abalone is predominantly eaten raw. This study investigated the effects of electron beam (e-beam) irradiation (1–10 kGy) on the inactivation of murine norovirus-1 (MNV-1), as a surrogate of human norovirus (NoV). The cell culture lysate of MNV-1 and abalone meat and viscera were irradiated with e-beam (1, 3, 5, 7, and 10 kGy). The titer of MNV-1 significantly (p < 0.05) decreased by 0.38–2.18, 0.31–1.45 and 0.41–1.56 log10 PFU/mL in the suspension, abalone meat, and abalone viscera, respectively, as the dose of e-beam irradiation increased. However, all Hunter colors, five sensory attributes and four textures parameter not significantly (p > 0.05) different in abalone after e-beam irradiation. D-values were calculated using first-order model and corresponded to 3.92, 6.26, and 5.23 kGy in the suspension, abalone meat, and abalone viscera, respectively. There were no significant changes in food qualities of pH, moisture contents and TBARS levels. Thus, e-beam can be used as an effective and useful non-thermal treatment to reduce MNV-1 in abalone. We suggest using a dose of e-beam irradiation >6.26 kGy to achieve 90% log10 reduction of MNV-1 in abalone without deleterious changes of food qualities.

A nonenveloped virus with a lipid envelope: hepatitis A virus as used in virus-reduction studies.

Recently, a quasi-lipid-enveloped (LE) form of the traditionally nonlipid-enveloped (NLE) hepatitis A virus (HAV) was described in human serum and cell culture-derived HAV stocks. This discovery challenges the understanding of HAV reduction in virus clearance studies of plasma products, which were performed under the premise of an NLE nature of this virus. Here, the presence of LE particles in HAV stocks used for reduction studies was verified, and the hypothesis that LE and NLE particles might contribute to the differential heat sensitivity of HAV variants during heat treatment of human serum albumin was evaluated. Cell culture lysates and supernatants of two cytopathic HAV variants, HM175/18f and HM175/24a, were characterized for their LE and NLE particle content by isopycnic gradient centrifugation. The obtained fractions were characterized for relative infectivity and then subjected to heat treatment (58.0 ± 1.0°C for 590 ± 10 minutes) in 12.5% human serum albumin to investigate their respective heat sensitivity. Preparations of the two HAV variants contained either LE particles (HM175/24a) or LE and NLE particles (HM175/18f) with equivalent specific infectivity. For HM175/18f, heat sensitivity of LE and NLE fractions did not differ significantly, and inactivation of the whole virus stock was identical to the NLE particle inactivation profile, whereas the HM175/24a variant was more heat sensitive. The results indicate that, in heat-treatment studies, the LE or NLE HAV phenotype is less important than the choice of HAV variant, and the most heat-resistant HM175/18f should be used.

Microbial detoxification of eleven food and feed contaminating trichothecene mycotoxins

BackgroundContamination of agricultural commodities with multiple trichothecene mycotoxins, produced by toxigenic Fusarium species, is a food safety issue, which greatly affects grain production and marketing worldwide. Importantly, exposure to multiple trichothecenes may increase toxicity in animals due to their synergistic and/or additive effects. To address the problem this study aimed to achieve a novel biological trait capable of detoxifying various food and feed contaminating trichothecenes under aerobic and anaerobic conditions and wide range of temperatures.ResultsA highly enriched microbial consortium (called DX100) capable of transforming eleven trichothecenes to significantly less toxic de-epoxy forms was achieved after prolonged incubation of soil microbial culture with 200 μg/mL deoxynivalenol (DON). DX100 demonstrated de-epoxidation activity under aerobic and anaerobic conditions, a greater range of temperatures and around neutral pH. The consortium contains 70% known and 30% unknown bacterial species, dominated by Stenotrophomonas species. Probably novel bacteria including strains of Stenotrophomonas and Alkaliphilus-Blautia species complex could be involved in aerobic and anaerobic de-epoxidation of trichothecenes, respectively. DX100 showed rapid and stable activity by de-epoxidizing 100% of 50 μg/mL deoxynivalenol at 48 h of incubation and retaining de-epoxidation ability after 100 subcultures in mineral salts broth (MSB). It was able to de-epoxidize high concentration of DON (500 μg/mL), and transformed ten more food contaminating trichothecenes into de-epoxy forms and/or other known/unknown compounds. Microbial de-epoxidation rate increased with increasing trichothecene concentrations in the broth media, suggesting that DX100 maintains a robust trichothecene detoxifying mechanism. Furthermore, the nature of microbial de-epoxidation reaction and inhibition of the reaction by sodium azide and the finding that bacterial cell culture lysate retained activity suggests that certain cytoplasmic reductases may be responsible for the de-epoxidation activity.ConclusionsThis study reports the enrichment procedure for obtaining an effective and stable microbial consortium DX100 capable of de-epoxidizing several food contaminating trichothecene mycotoxins. DX100, which has de-epoxidation ability under wide range of conditions, represents a unique enzymatic source which has great industrial potential for reducing contamination of foods/feeds with multiple trichothecenes, and minimizing their synergistic/additive cytotoxic effects on consumer health.

Label-Free Isothermal Amplification Assay for Specific and Highly Sensitive Colorimetric miRNA Detection.

We describe a new method for the detection of miRNA in biological samples. This technology is based on the isothermal nicking enzyme amplification reaction and subsequent hybridization of the amplification product with gold nanoparticles and magnetic microparticles (barcode system) to achieve naked-eye colorimetric detection. This platform was used to detect a specific miRNA (miRNA-10b) associated with breast cancer, and attomolar sensitivity was demonstrated. The assay was validated in cell culture lysates from breast cancer cells and in serum from a mouse model of breast cancer.

Validation of Metabolic Alterations in Microscale Cell Culture Lysates Using Hydrophilic Interaction Liquid Chromatography (HILIC)-Tandem Mass Spectrometry-Based Metabolomics.

By standard convention, in order to increase the efficacy of metabolite detection from cell culture lysates, metabolite extracts from a large quantity of cells are utilized for multiple reaction monitoring-based metabolomic studies. Metabolomics from a small number of cell extracts offers a potential economical alternative to increased cell numbers, in turn increasing the utility of cell culture-based metabolomics. However, the effect of reduced cell numbers on targeted metabolomic profiling is relatively unstudied. Considering the limited knowledge available of the feasibility and accuracy of microscale cell culture metabolomics, the present study analyzes differences in metabolomic profiles of different cell numbers of three pancreatic cancer cell lines. Specifically, it examines the effects of reduced cell numbers on metabolite profiles by obtaining extracts either directly from microscale culture plates or through serial dilution of increased numbers of cellular metabolite extracts. Our results indicate reduced cell numbers only modestly affect the number of metabolites detected (93% of metabolites detected in cell numbers as low as 104 cells and 97% for 105 cells), independent of the method used to obtain the cells. However, metabolite peak intensities were differentially affected by the reduced cell numbers, with some peak intensities inversely proportional to the cell numbers. To help eliminate such potential inverse relationships, peak intensities for increased cell numbers were excluded from the comparative analysis. Overall, metabolite profiles from microscale culture plates were observed to differ from the serial dilution samples, which may be attributable to the medium-to-cell-number ratios. Finally, findings identify perturbations in metabolomic profiling for cellular extracts from reduced cell numbers, which offer future applications in microscale metabolomic evaluations.

Clinical correlation of extensive-stage small-cell lung cancer genomics

Genomic studies in small-cell lung cancer (SCLC) lag far behind those carried out in nonsmall-cell lung cancer (NSCLC). To date, most SCLC studies have evaluated patients with surgically resectable disease. Here we sought to evaluate the genomic mutation spectrum of 'every-day' SCLC patient tumors with extensive stage disease (ES-SCLC) and to correlate mutations with the main clinical outcomes of response to chemotherapy, progression-free (PFS) and overall (OS) survival. A total of 50 SCLC patient tumors were examined in this study; targeted exome sequencing was obtained on 42 patients and whole-exome sequencing on 8 patients. Mutated genes were correlated with clinical outcomes using Kaplan-Meier methods (PFS, OS) and logistic regression (chemo-response). RB1 protein expression was detected by either western blotting of cultured cell lysates or immunohistochemistry of tumor specimens. In all, 39 patients had ES-SCLC; 15 patients had either primary refractory/resistant disease and 21 patients had sensitive disease. The two most frequently mutated genes were TP53 (86%) and RB1 (58%); other frequently mutated genes (>10% patients) were involved in epigenetic regulation as well as the mTOR pathway. We identified a number of low-frequency, targetable mutations, including RICTOR, FGFR1, KIT, PTCH1 and RET. Using multivariate analysis, RB1 was the only significant factor (P = 0.038) in predicting response to first-line chemotherapy, with an odds ratio of 5.58 comparing mutant RB1 with wild-type. Patients with mutant RB1 had both better OS (11.7 versus 9.1 months P = 0.04) and PFS (11.2 versus 8.6 months, P = 0.06) compared with patients with wild-type RB1. Interestingly, ∼25% of SCLC cell lines and tumor specimens expressed RB1 protein, possibly representing the subgroup with wild-type RB1. We found that SCLC tumors harboring no mutation in RB1 had a poor response to chemotherapy.

Soluble CD163 modulates cytokine production by peripheral blood mononuclear cells stimulated by Dermatophagoides pteronyssinus allergens in vitro

PurposeThe CD163 is a scavenger receptor expressed exclusively on monocytes/macrophages which has been shown to exert anti-inflammatory effects. The aim of this study was to evaluate the effect of exogenous sCD163 on production of selected cytokines by peripheral blood mononuclear cells (PBMC) of house dust mite allergic patients (AAPs) stimulated in vitro with Dermatophagoides pteronyssinus (Dp) allergens. Patients and methodsThe study was performed in 24 AAPs and 12 healthy control subjects (HCs). Peripheral blood mononuclear cells were cultured for up to 144h (T144) in the presence of Dp extract with or without sCD163. Concentration of interleukin (IL) – 10, IL-13 and transforming growth factor beta (TGF-β) was evaluated in the cell culture supernatants using ELISA. Expression of the selected cytokines was evaluated in cell culture lysates using Taqman-based real time polymerase chain reaction (RT-PCR). ResultsDp-stimulated PBMC from AAPs released more IL-10 and IL-13 than those from HCs. The greatest up-regulation of IL-10 expression was seen at T6, while that of IL-13 was delayed. Soluble CD163 augmented production of IL-10 in response to Dp stimulation. No significant effect of sCD163 on production of IL-13 and IL-10 by PBMC of HCs could be demonstrated. ConclusionsIn AAPs sCD163 modulates the immune response to Dp allergens potentiating anti-inflammatory, homeostatic mechanisms.

Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector

With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development.