Peripheral Blood Cell Cultures Research Articles

Short-chain fatty acids induced lung tumor cell death and increased peripheral blood CD4+ T cells in NSCLC and control patients ex vivo.

Despite therapy advances, one of the leading causes of cancer deaths still remains lung cancer. To improve current treatments or prevent non-small cell lung cancer (NSCLC), the role of the nutrition in cancer onset and progression needs to be understood in more detail. While in colorectal cancer, the influence of local microbiota derived SCFAs have been well investigated, the influence of SCFA on lung cancer cells via peripheral blood immune system should be investigated more deeply. In this respect, nutrients absorbed via the gut might affect the tumor microenvironment (TME) and thus play an important role in tumor cell growth. This study focuses on the impact of the short-chain fatty acid (SCFA) Sodium Butyrate (SB), on lung cancer cell survival. We previously described a pro-tumoral role of glucose on A549 lung adenocarcinoma cell line. In this study, we wanted to know if SB would counteract the effect of glucose and thus cultured A549 and H520 in vitro with and without SB in the presence or absence of glucose and investigated how the treatment with SB affects the survival of lung cancer cells and its influence on immune cells fighting against lung cancer. In this study, we performed cell culture experiments with A549, H520 and NSCLC-patient-derived epithelial cells under different SB levels. To investigate the influence on the immune system, we performed in vitro culture of peripheral mononuclear blood cells (PBMC) from control, smoker and lung cancer patients with increasing SB concentrations. To investigate the effect of SB on lung tumor cells, we first analyzed the effect of 6 different concentrations of SB on A549 cells at 48 and 72 hours cell culture. Here we found that, SB treatment reduced lung cancer cell survival in a concentration dependent manner. We next focused our deeper analysis on the two concentrations, which caused the maximal reduction in cell survival. Here, we observed that SB led to cell cycle arrest and induced early apoptosis in A549 lung cancer cells. The expression of cell cycle regulatory proteins and A549 lung cancer stem cell markers (CD90) was induced. Additionally, this study explored the role of interferon-gamma (IFN-γ) and its receptor (IFN-γ-R1) in combination with SB treatment, revealing that, although IFN-γ-R1 expression was increased, IFN-γ did not affect the efficacy of SB in reducing tumor cell viability. Furthermore, we examined the effects of SB on immune cells, specifically CD8+ T cells and natural killer (NK) cells from healthy individuals, smokers, and NSCLC patients. SB treatment resulted in a decreased production of IFN-γ and granzyme B in CD8+ T cells and NK cells. Moreover, SB induced IFN-γ-R1 in NK cells and CD4+ T cells in the absence of glucose both in PBMCs from controls and NSCLC subjects. Overall, this study highlights the potential of SB in inhibiting lung cancer cell growth, triggering apoptosis, inducing cell cycle arrest, and modulating immune responses by activating peripheral blood CD4+ T cells while selectively inducing IFN-γ-R1 in NK cells in peripheral blood and inhibiting peripheral blood CD8+ T cells and NK cells. Thus, understanding the mechanisms of action of SB in the TME and its influence on the immune system provide valuable insights of potentially considering SB as a candidate for adjunctive therapies in NSCLC.

Research of the Potential Radioprotective Activity of Trimethyl Glycine and N-Acetyl-L-Cysteine with Quantitative Analysis Using Real-Time Polymerase Chain Reaction (QRT-PCR)

The irradiation exposure could affect the cellular genome and specifically target genes, such as TP53 gene. The TP53 gene is known as a “guardian of the genome” and is a tumor suppressor gene. Its role is to keep the cell of malignant transformation. After ionizing radiation exposure, the TP53 could mutate and the cell could continue to active proliferation (tumor cellular transformation). It has been noticed that different type of substances could have radioprotective effect and to protect the cell of malignant transformation, apoptosis, genome injuries or necrosis. Some of the natural metabolites that are proven antioxidants, show significant ability of radiation protection of the cells. The aim of that study is to analyze the possible radioprotection ability of two origin metabolites (amino acids)–trimethyl glycine (betaine) and N-acetyl-L-cysteine, applied together and in combination, first before irradiation (preventive treatment) and then 2 hours after irradiation (as a therapeutic agents). The research work is done as invitro analysis to peripheral blood cell cultures. Conclusion: The conclusion of the study is that both amino acids showed good radiation protection activity after in-vitro performed analysis. The most significant results showed the combination of both natural metabolites.

Influence of alpha-glutamil-tryptophan on the background and induced activity of factors of adaptive immunity for prevention

Background. During the ongoing COVID-19 pandemic and in the season of rising incidence of other respiratory infections, it is relevant to use preventive measures of non-specific prophylaxis. Synthetic peptides are widely considered as a tool. The representative of this group is the synthetic analogue of thymus regulatory peptides Thymogen, which has been used in Russia for more than 20 years in the treatment of acute and chronic infection diseases.The aim of the study. To evaluate the effect of Thymogen, a dosed nasal spray, on induced parameters of the immune system during prophylactic use in healthy volunteers.Materials and methods. Twenty healthy volunteers received Thymogen nasal dosed spray (JSC “Cytomed”, Russia) at a dose of 25 μg twice a day for 10 days. A comparative assessment of immunological parameters was carried out in dynamics: before the start of therapy, on days 6 and 11 of taking the drug and 14 days after the end of the course. Clinical observation was carried out from day 1 to day 11, registration of adverse events – the entire period of the study for 24 days. The first day was considered the day the drug was started.Results. In the course of the work, according to the data of immunological examination, a statistically significant increase in the virus-induced production of interferon alpha (INF-α) by a culture of peripheral blood cells was revealed. The growth rate was recorded on day 11 of taking Thymogen and persisted for 14 days after the end of the course. Significant differences in the dynamics of bactericidal and phagocytic activity of neutrophils, serum α- and γ-interferon were not obtained.Conclusion. The use of Thymogen spray at a dose of 25 μg for 10 days was safe and contributed to a significant induction of interferon-alpha in response to exposure to a viral pathogen, which allows us to recommend the drug for prophylactic use during the period of rising incidence of acute respiratory diseases.

3D microenvironment attenuates simulated microgravity-mediated changes in T cell transcriptome.

Human space travel and exploration are of interest to both the industrial and scientific community. However, there are many adverse effects of spaceflight on human physiology. In particular, there is a lack of understanding of the extent to which microgravity affects the immune system. T cells, key players of the adaptive immune system and long-term immunity, are present not only in blood circulation but also reside within the tissue. As of yet, studies investigating the effects of microgravity on T cells are limited to peripheral blood or traditional 2D cell culture that recapitulates circulating blood. To better mimic interstitial tissue, 3D cell culture has been well established for physiologically and pathologically relevant models. In this work, we utilize 2D cell culture and 3D collagen matrices to gain an understanding of how simulated microgravity, using a random positioning machine, affects both circulating and tissue-resident T cells. T cells were studied in both resting and activated stages. We found that 3D cell culture attenuates the effects of simulated microgravity on the T cells transcriptome and nuclear irregularities compared to 2D cell culture. Interestingly, simulated microgravity appears to have less effect on activated T cells compared to those in the resting stage. Overall, our work provides novel insights into the effects of simulated microgravity on circulating and tissue-resident T cells which could provide benefits for the health of space travellers.

Concentration of anti-inflammatory cytokines in supernatants of peripheral blood cell cultures in children with autoimmune and infectious diseases

Cytokines belong to the class of signaling molecules, being involved into regulation of proliferation, differentiation and effector functions of immunocompetent cells. The ratio of pro- and anti-inflammatory cytokines is important for development of any inflammatory process. IL-1ra, IL-4, IL-10 are among the most important anti-inflammatory cytokines. Their function is to limit and suppress immune response in inflammatory processes of any etiology. In this respect, the aim of this study was to evaluate production of the anti-inflammatory cytokines IL-1ra, IL-4 and IL-10 by peripheral blood cells in children with autoimmune and infectious diseases. Patients and methods: Pediatric parients (2-17 years old) participated in the study including those with juvenile idiopathic arthritis (n = 101); unspecified reactive arthropathy (n = 24); systemic lupus erythematosus (SLE, n = 14); chronic viral hepatitis C (n = 24). 33 healthy children (n = 33) comprised the control group. Heparinized blood samples were diluted with glutamine-containing medium RPMI-1640. Control samples were not treated by any stimulants, and the stimulated samples were supplied with phytohemagglutinin (20 mkg/ml). The samples of diluted blood were incubated for 24 hours (37 C, 5% CO2). The supernates were frozen once. The concentrations of IL-1ra, IL-4 and IL-10 in these cell supernatants were determined by ELISA technique (Vector-Best, Russia). Results: It was found that the spontaneous production of anti-inflammatory cytokines (IL-1ra, IL-4 and IL-10) didnt differ, or was lower in the groups of patients with SLE, juvenile idiopathic arthritis and hepatitis C if compared with control group. SLE is an autoimmune disease, whereas juvenile arthritis is of mixed autoimmune-autoinflammatory etiology. Chronic hepatitis C is a viral disease, but autoimmune responses may manifest at the chronic stage of the disorder. Decreased production of anti-inflammatory cytokines could be an evidence for autoimmune mechanisms of these diseases, despite their different etiology. More intensive spontaneous production of IL-1ra and IL-10 in children with unspecified reactive arthropathy may suggest some compensatory reactions which inhibit development of inflammation in this disorder. IL-1ra, IL-4 and IL-10 production in stimulated cultures didnt differ between all groups of the patients, or it was lower in comparison with healthy children. Decrease cytokine production in groups of children with different diseases suggests exhausted functional reserve of immunocompetent cells caused by their chronic activation.

Recipient Tregs: Can They Be Exploited for Successful Hematopoietic Stem Cell Transplant Outcomes?

Human and mouse CD4+FoxP3+ T cells (Tregs) comprise non-redundant regulatory compartments which maintain self-tolerance and have been found to be of potential therapeutic usefulness in autoimmune disorders and transplants including allogeneic hematopoietic stem cell transplantation (allo-HSCT). There is substantial literature interrogating the application of donor derived Tregs for the prevention of graft versus host disease (GVHD). This Mini-Review will focus on the recipient’s Tregs which persist post-transplant. Although treatment in patients with low dose IL-2 months post-HSCT are encouraging, manipulating Tregs in recipients early post-transplant is challenging, in part likely an indirect consequence of damage to the microenvironment required to support Treg expansion of which little is understood. This review will discuss the potential for manipulating recipient Tregs in vivo prior to and after HSCT (fusion proteins, mAbs). Strategies that would circumvent donor/recipient peripheral blood harvest, cell culture and ex-vivo Treg expansion will be considered for the translational application of Tregs to improve HSCT outcomes.

Evaluation of in vitro Activation of Bovine Endometrial and Vaginal Epithelial and Blood Mononuclear Cells to Produce Nitric Oxide in Response to Mycoplasma bovis, Mycoplasma bovigenitalium and Ureaplasma diversum

Abstract Genital mycoplasmosis is a condition present in bovine production systems, and the most important agents involved are Mycoplasma bovis, Mycoplasma bovigenitalium and Ureaplasma diversum. Some aspects of their pathogenesis remain unclear. This study was designed in order to evaluate their ability to stimulate mononuclear cells from the endometrium, vagina and peripheral blood of cycling and healthy cows to produce nitric oxide (NO). Cellular cultures of endometrial, vaginal and peripheral blood cells from 33 healthy cows were cultivated with Mycoplasma bovis, Mycoplasma bovigenitalium and Ureaplasma diversum originated from the 4th passage in culture broth and the NO production was measured by the Greiss reaction. Confirmation of the presence of mononuclear cells and of the agents during and after the NO assay was done by Giemsa stained smears and further cultivation and detection by PCR reaction. Mononuclear cells from all samples produced NO. Mycoplasma bovigenitalium stimulated higher NO production than the others (p<0.05). Endometrial cells produced less NO than vaginal or blood cultured cells. In conclusion, it seems that Mycoplasma bovis, Mycoplasma bovigenitalium, and Ureaplasma diversum are able to activate mononuclear cells and induce the production of NO, thus suggesting that this pathway is elicited in response to the primary infection by these agents. More studies are necessary to verify why these agents remain in the bovine reproductive tract for long periods and how they reassume deleterious effects.

ASSESSMENT OF THE CYTOGENETIC CONSEQUENCES OF THE IMPACT OF UNUTILIZED AND BANNED TO USE PESTICIDES ON THE SHEEP ORGANISM

In the territories of those farms in the Almaty region, where previously engaged in vegetable growing, fruit growing and tobacco growing are old, abandoned warehouse where accumulated considerable amount unutilized and banned pesticides. These warehouses are located directly on the territories of settlements. There is no sanitary zone around them. The chemical constituents of non-utilized pesticides constantly pollute the environment and with the products of animal and vegetable origin produced in the same settlements, enter the body. The article is devoted to the cytogenetic assessment of the genotoxicity of unused and forbidden to use pesticides on the body of sheep. Animals are bred on pasture plots and kept in the settlements of Beskaynar, Kyzylkairat and Taukaraturyk, where old warehouses with pesticide. A cytogenetic analysis of chromosome preparations prepared from an in vitro culture of peripheral blood cells from 30 sheep was performed. The level of cells with genomic mutations in animals from three experimental sites was 2.6 2.96 and 2.75 times higher, respectively, in comparison with the corresponding indices of control animals. An analysis of the constituent genomic cell mutations in the blood system of these animals showed that cells with hyperdiploid sets of chromosomes were identified in 17 animals with a frequency from 0.67% to 2.88% of the analyzed metaphase cells. Polyploid cells were found in all animals, with an average frequency of 7.93 ± 1.21% (3484 cells). The level of cells with chromosomal aberrations in experimental animals is 6.25, 7.40 and 5.66 times higher than in control animals. Analysis of the level of cytogenetic instability in animal cells showed that in the blood system of experimental animals, the genotoxicity of unused and forbidden pesticides is detected due to an increase in the proportion of cells with hyperdiploid, polyploid sets of chromosomes and cells with chromosome aberrations. Key words: pesticides that are not utilized and banned to use, sheep, in vitro lymphocyte culture, genomic mutations, chromosomal aberrations.

IL-1β and TNFα measured in supernatants of peripheral blood cell cultures from children with autoimmune and infectious pathology

Cytokines are small-molecular weight proteins involved in regulating inflammation, proliferation and differentiation of immunocompetent cells, as well as effector functions. IL-1β and TNFα are among the most powerful inducers of the inflammatory response. Production of proinflammatory cytokines comprises normal response to infection and represents an arm of various autoimmune disease pathogenesis. It is worth comparing cytokine production in autoimmune and infectious diseases to determine features of cytokine profile. The aim of the study was to evaluate proinflammatory cytokines IL-1β and TNFα produced by blood cells in children with autoimmune and infectious diseases. 194 children, aged 2-17 years, were examined: 99 patients with juvenile idiopathic arthritis; 26 patients with unspecified reactive arthropathy; 14 children with systemic lupus erythematosus; 24 children with chronic viral hepatitis C; 33 healthy children. Heparinized blood samples were diluted with a glutamine-containing culture medium RPMI-1640, samples with/without phytohemagglutinin stimulation were prepared as well. Samples of diluted blood were incubated for 24 hours (37 °C, 5% CO2). The concentrations of IL-1β and TNFα in the cell culture supernatants were determined by ELISA. It was found that groups of patients with rheumatic diseases (systemic lupus erythematosus, juvenile idiopathic arthritis and unspecified reactive arthropathy) were featured with spontaneous production of IL-1β and TNFα at higher level than in control, and the stimulated synthesis of IL-1β was lower. In patients with chronic viral hepatitis C, the spontaneous concentration IL-1β and TNFα and the stimulated concentration of IL-1β did not differ those ones found in healthy children. Stimulated TNFα production in patients with juvenile idiopathic arthritis, unspecified reactive arthropathy, and hepatitis C was significantly higher than in control group. More intensive spontaneous production IL-1β and TNFα in groups of patients with rheumatic diseases indicates previous activation of immunocompetent cells. Decreased stimulated IL-1β production in groups with various diseases points at exhaustion of immunocompetent cell functional reserve due to chronic activation.

Blood-based inflammation biomarkers of neurocognitive impairment in people living with HIV.

Inflammation in people living with HIV (PLWH) correlates with severity of HIV-associated neurocognitive disorders. The objective of this study is to identify blood-based markers of neurocognitive function in a demographic balanced cohort of PLWH. Seven neurocognitive domains were evaluated in 121 seropositive Black/African American, Non-Hispanic White, and White Hispanic men and women using computerized assessments. Associations among standardized neurocognitive function and HIV-related parameters, relevant sociodemographic variables, and inflammation-associated cytokines measured in plasma and cellular supernatants were examined using multivariate and univariate regression models. Outlier and covariate analyses were used to identify and normalize for education level, CD4 T cell count, viral load, CNS and drug abuse comorbidities, which could influence biomarker and neurocognitive function associations. Plasma levels of chemokine (C-C motif) ligand (CCL) 8 significantly associated with memory, complex attention, cognitive flexibility, psychomotor speed, executive function, and processing speed. Plasma tissue inhibitor of metalloproteinases 1 associated with the aforementioned domains except memory and processing speed. In addition, plasma interleukin-23 significantly associated with processing speed and executive function. Analysis of peripheral blood cell culture supernatants revealed no significant markers for neurocognitive function. In this cohort, CD4 T cell count and education level also significantly associated with neurocognitive function. All identified inflammatory biomarkers demonstrated a negative correlation to neurocognitive function. These cytokines have known connections to HIV pathophysiology and are potential biomarkers for neurocognitive function in PLWH with promising clinical applications.

Three patients with 46,X,inv(Y)(p11.2q11.2)pat/45,X and their pedigree analysis.

The present study aimed to perform chromosome examination and pedigree analysis on three patients with semen abnormality who had undergone in vitro fertilization–embryo transfer (IVF‐ET). Peripheral blood cell culture and chromosome karyotyping were performed on 4,200 individuals who had undergone chromosome examination. Among them, 155 pregnant women who had successfully conceived were subjected to amniotic cell culture and chromosome karyotyping and those with abnormal chromosome karyotype were further subjected to C‐banding and whole‐genome sequencing. Mosaicism for a 46,X,inv(Y)(p11.2q11.2)pat/45,X karyotype was identified in the probands and immediate adult male relatives. The incidence of this mosaicism in the study population was only 0.07% (3/4,200), which is reported for the first time. For the proband of pedigree A, the results of whole‐genome sequencing and other tests were normal, and the chromosome karyotype of IVF fetuses was 46,X,inv(Y)(p11.2q11.2)pat. All the male members of three pedigrees have normal phenotypes, with no features of Turner's syndrome (45,X) or hermaphroditism (45,X/46,XY), suggesting that the inverted Y chromosome is extremely unstable and particularly susceptible to loss in somatic cells. So we speculate this karyotype may be a unique type of inverted Y chromosome in somatic cells.

Low Mononuclear Cell IL-18 and IL-27 Response in Children: Susceptibility to Tuberculosis Infection after Contact

Background Identification of the immune response against tuberculosis is vital to develop new diagnostic and therapeutic modalities. The objective of this study was to determine IL (interleukin)-18 and IL-27 responses of peripheral blood mononuclear cells to early secreted antigen (ESAT-6) and culture filtrate protein-10 (CFP-10) stimulation in children with a (+) or (−) tuberculin skin test (TST) with in-house tuberculosis contact. Methods We enrolled 40 children aged 1 to 5 years who had an in-house contact with a tuberculous adult. Blood samples were obtained from all children for QuantiFERON tuberculosis (TB) gold in tube (QFT-GIT), and peripheral blood mononuclear blood cell culture tests. The subjects were grouped as TST (−) QFT-GIT (−), TST (+) QFT-GIT (−), and TST (+) QFT-GIT (+). Supernatant of peripheral blood mononuclear cell culture was separated with and without stimulation of ESAT-6 and CFP-10, and IL-18 and IL-27 levels were measured with enzyme linked immunoassay (ELISA) test. Results The study group included 22 boys and 18 girls with mean age 4.25 ± 0.9 years. IL-18 and IL-27 levels were statistically significant in ESAT-6/CFP-10-stimulated supernatants of peripheral blood mononuclear cell (PBMC) samples among the three groups (p = 0.000, p = 0.007, respectively). IL-18 levels between the TST (−) QFT-GIT (−) and TST (+) QFT-GIT (+) groups were significantly different (p = 0.026). Both IL-18 and IL-27 levels were significantly different between ESAT-6/CFP-10 stimulated PBMC supernatants of TST (−) QFT-GIT (−) and TST (+) QFT-GIT (−) groups (p = 0.000, p = 0.003, respectively). Conclusion Low IL-18 and IL-27 responses of peripheral blood mononuclear cells in children with Bacillus Calmette–Guérin (BCG) vaccine may play a role in Mycobacterium tuberculosis infection after in-house contact.

Aspects of the immune response in sheep experimentally co-infected with Corynebacterium pseudotuberculosis and Haemonchus contortus

The present work aimed to evaluate the possible negative regulation induced by Th2 mechanisms, unleashed by the helminth Haemonchus contortus, on the response by cells of the Th1 profile, important to contain the Corynebacterium pseudotuberculosis cocobacillus infection, in the concomitant ovine infection by these two parasites. For this, the Th1 response was evaluated in two different experimental infection conditions, using the in vitro production of interferon, which is the most typical cytokine of its profile, from cultures of peripheral blood cells of ovine animals co-infected with these two parasites, compared to their production, under the same conditions, in animals only infected with the said bacterium and using as stimulus two antigen preparations with different degrees of purity. The results evidenced a tendency of reduction in the production of this cytokine in the co-infected animals in relation to those infected with Corynebacterium pseudotuberculosis alone, in the two experimental protocols. As for the response by IgG class specific antibodies against this bacterium, evaluated by ELISA and western blot, no differences were observed between groups infected only with bacillus or with double infection.

Interferon-gamma release assay performance in northeastern Brazil: influence of the IFNG + 874 A>T polymorphism

IntroductionLatent tuberculosis infection diagnosis based on the release of interferon-gamma in cultures of peripheral blood cells stimulated with Mycobacterium tuberculosis antigens has replaced the tuberculin skin test in many countries with low tuberculosis prevalence. The IFN-γ production can be influenced by genetic polymorphisms, of which the IFNG+874 (rs62559044) locus is the most studied. We investigated the possible influence of the IFNG+874 A/T polymorphism on interferon-gamma test performance. MethodsPatients diagnosed with pulmonary tuberculosis (75), volunteers with positive tuberculin skin test (70) and healthy volunteers with negative tuberculin skin test and no history of contact with tuberculosis (57) were evaluated regarding the IFNG+874 genotype and the IFN-γ levels in whole blood cultures performed using an interferon-gamma commercial kit (QuantiFERON-TB® Gold In-Tube). ResultsIFN-γ production was not influenced by the IFNG+874 genotype, regardless of antigen or mitogen-based stimulation, which suggests that other genes may influence IFN-γ production in response to mycobacteria. The IFNG+874 polymorphism was found to exert no influence over QFT-IT test sensitivity in our study. ConclusionsThe IFNG+874 polymorphism was not shown to influence QuantiFERON-TB® Gold In-Tube test performance in an admixed population from northeastern Brazil.

Correlation of CD34+ Hematopoietic Stem Cells and CFU in Peripheral Blood Apheresis Products in Patients with Malignant Lymphoproliferative Diseases Before and After Cryopreservation Prior to auto-HSCT

Aim. To establish correlation between CD34+ autologous hematopoietic stem cell (HSC) count and colony-forming units (CFU) in the same peripheral blood apheresis product samples before and after cryopreservation in multiple myeloma and lymphoma patients, and to assess clinical value of these parameters. Materials & Methods. Cell samples of peripheral blood cyta-pheresis product and cell cultures were studied before and after cryopreservation in 32 multiple myeloma and 25 lymphoma patients who underwent autologous HSC transplantation. The material was analyzed using culture technique and flow cytometry. Results. The paper provides information on the relationship between CD34+ HSC count obtained by flow cytometry, and CFU in cell culture obtained by cytapheresis of the same peripheral blood samples. A direct correlation was confirmed between CD34+ count and all the CFUs before and after cryopreservation in lymphoma patients. Correlation between CD34+ count and granulocyte-macrophage CFUs was revealed in multiple myeloma and lymphoma patients before cryopreservation. Conclusion. The parameter of colony-forming capacity used for the assessment of the functional HSC was shown to be equally reliable criterion for condition evaluation of autotransplant proliferative pool than CD34+ cells. Both methods should be applied for qualitative and quantitative evaluation of an autotransplant for multiple myeloma and lymphoma patients.

Abstract B095: Multiplexed detection of RNA and proteins on the nCounter® platform with low sample input protocol

Abstract As our understanding of the immune responses to cancer continues to grow, the need to extract greater amounts of information from ever smaller sample sizes increases. One of the biggest challenges facing the field is to develop a comprehensive understanding of how the immune system responds to a tumor, and multi-omic profiling (DNA, RNA, and protein) is crucial to building a holistic model of tumor immunity. The NanoString nCounter platform has become an important tool in quantifying transcriptional responses from a wide variety of sample types by enabling direct digital counting of up to 800 targets from a single sample using nucleic acid probes that directly hybridize to the RNA sequence of interest and then are quantified with optical barcoding technology. The platform has now been extended to permit quantification of proteins at the same time using primary antibodies conjugated to DNA oligos that hybridize with the barcodes. We have developed new technology that enables characterization of up to 30 proteins and 770 RNA transcripts in key immuno-oncology pathways from a very low amount of starting material - as few as 50,000 cells. NanoString has previously developed the nCounter Vantage RNA:Protein Immune Profiling Panel which allows digital counting of extracellular proteins which facilitates quantitation of multiple immune cell populations and provides information about their activation status. We have now applied this technology to detect intracellular and secreted proteins as well in the new nCounter Vantage RNA:Protein Immune Signaling panel. This panel is able to detect key transcription factors, signaling molecules, and secreted proteins from peripheral blood mononuclear cells (PBMC), dissociated cells, or cell culture. Additionally, we have recently developed a universal cell capture technology that utilizes anti-β2M antibody coupled to magnetic beads to pull down nucleated cells from a large starting volume. As proof of concept, PBMC from a healthy donor were treated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, TNFα, or IFNγ and RNA and protein were characterized with both the Immune Profiling and Immune Signaling panels. nCounter protein detection compared favorably when validated with flow cytometry, and the additional information imparted by simultaneous profiling of the RNA transcriptome enabled mapping of signaling pathways activated by treatment. Furthermore, RNA and protein counts from the low input protocol were representative of counts obtained from higher cell inputs, indicating that no loss or skewing of data resulted from reducing the starting material. This advance in multi-analyte, multiplexed digital molecular profiling will accelerate immuno-oncology research by reducing sample size requirements and may enable the discovery and development of novel immunotherapies and their associated companion diagnostics. Citation Format: Sarah Warren, Gary Geiss, Brian Burditt, Qian Mei, Alan Huang, Maribeth Eagen, Eduardo Ignacio, Dwayne Dunaway, Lucas Dennis, Joseph Beechem. Multiplexed detection of RNA and proteins on the nCounter® platform with low sample input protocol [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B095.

Role of myeloid early endothelial progenitor cells in bone formation and osteoclast differentiation in tissue construct based on hydroxyapatite poly(ester-urethane) scaffolds.

Engineering of a vascularized bone construct is a highly challenging task which needs to take into account the impact of different components on the bone regeneration process. Bone repair influencing factors in such constructs range from the material properties and scaffold design, to the interaction of different cell types contributing to bone formation and remodeling or neovascularization, respectively. In this context, early endothelial progenitor cells (EPC), mononuclear cells isolated from the peripheral blood, express the endothelial marker CD31 but also a series of myeloid markers and have been shown to support the formation of vessel-like structures. These cells are also characterized by a highly adaptable phenotype influenced by other cells creating an instructive niche. The present study was designed to investigate the impact of EPC on bone formation or remodeling using a co-culture system of outgrowth endothelial cells, mature endothelial cells isolated from the peripheral blood cell cultures, and mesenchymal stem cells grown on hydroxyapatite poly(ester-urethane) scaffolds. The formation of vessel-like structures in these constructs was shown by CLSM and immunohistochemistry and further evaluated by real time RT-PCR. Osteogenic differentiation in these constructs was investigated by von Kossa, Alizarin Red, and real time PCR. Data indicated that osteogenic differentiation occurred within the constructs after 14 days of culture but without a direct influence by EPC in this process. Finally, although we observed a series of osteoclast related makers in the constructs when EPC were included, no indications for an increased osteoclast-like activity, which might lead to increased bone resorption, were observed. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1922-1932, 2016.

Acetylated Trivalent Mannobioses: Chemical Modification, Structural Elucidation, and Biological Evaluation.

People suffering from allergies can be treated with repeated injections of increasing amounts of a specific allergen. This type of specific immunotherapy is currently the only way to treat the underlying pathological immune response associated with an allergy. The approach can afford long-lasting protection, but the process takes 3-5 years, can produce allergic reactions, and in severe cases treatment is often aborted due to anaphylaxis. However, treatment can be optimized with the use of specific adjuvants that modify the immune response, its duration, and that increase the production of the correct type of antibodies. In the pursuit of such adjuvants, two new trivalent acetylated β-(1→2)-linked mannobioses based on a previously discovered lead molecule were prepared. The new molecules, along with the previously developed lead, were investigated by rigorous NMR and molecular modeling experiments in order to elucidate their behavior and preferred conformations in solution. Furthermore, the molecules were subjected to a biological investigation in which their immunostimulatory properties were evaluated by assessing their effect on the production of TH 2-type cytokine interleukin-4 (IL-4) and Treg pro-inflammatory cytokine tumor necrosis factor (TNF). Treatment of peripheral mononuclear blood cell cultures from patients suffering from birch allergy with birch allergen Bet v induced a strong IL-4 response, whereas the same treatment together with the trivalent acetylated mannobioses caused significant suppression of the induced IL-4.

Chromosome fragility in river buffalo cows exposed to dioxins

Fifty river buffalo (Bubalus bubalis, 2n = 50) cows reared in two different provinces of Campania (southern Italy) underwent cytogenetic investigations to ascertain possible differences in their chromosome stability. One group (Caserta province) was under legal sequestration due to the presence in the milk mass of higher mean values of dioxins [21.79pg/g of fat as sum of polychloro-dibenzo-dioxins (PCDDs), polychloro-dibenzo-furans (PCDFs) and dioxin-like polychlorobiphenyls (DL-PCBs)] than both those permitted (6.0pg/g of fat as WHO-TEQ) and those (1.3pg/g of fat as WHO-TEQ) observed in the control group raised in Salerno province. Two types of peripheral blood cell cultures were performed: without (normal cultures for the chromosome abnormality (CA) test: chromatid breaks, chromosome breaks, fragments) and with the addition of BrdU for the sister chromatid exchange (SCE) test). The CA test revealed a significantly (P < 0.01) higher chromosome fragility in the exposed cows compared to the control. Indeed, mean values of CA/cell were 1.26 ± 1.15 in exposed cows and 0.37 ± 0.71 in the control. Mean SCE was higher in exposed cows (8.50 ± 3.35) than that (8.29 ± 3.51) found in the control but the difference was not significant. Comparison within the same group of cows at first (FL) and multiple (ML) lactations revealed significantly (P < 0.01) higher mean values of CA/cell in exposed ML-cows vs FL-cows while no statistical differences were found between ML-cows and FL-cows in the control farm. By contrast, significantly (P < 0.01) higher mean values of SCE were found in both groups of FL-cows versus ML-cows. Comparisons with other previous studied species (sheep and cattle) were also performed.

Nanoparticles Containing siRNA to Silence CD4 and CCR5 Reduce Expression of These Receptors and Inhibit HIV-1 Infection in Human Female Reproductive Tract Tissue Explants

Human Immunodeficiency Virus-type 1 (HIV- 1) binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA) transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV- 1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA) sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α), a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.