Cells In Lymphocyte Culture Research Articles

Effect of interferon-alpha-containing drugs on innate immunity reactions and population and subpopulation composition of peripheral blood lymphocytes

Introduction. Interferon-containing drugs are frequently used, but their effect on the innate immune cells response and adaptive immunity parameters is not well known.Aim. To investigate the effect of interferon-alpha-containing drugs on the production of neutrophil extracellular traps, the population and subpopulation composition of peripheral blood lymphocytes in cell culture conditions.Materials and methods. We used peripheral blood from 12 healthy donors and 6 patients with acute inflammation, which was used as a neutrophil and lymphocyte source. Neutrophil extracellular traps were induced by two methods: either spontaneously, without stimulation, or by lipopolysaccharides.Results and discussions. The investigation of innate immunity responses showed that neutrophils from healthy donors demonstrated a slight spontaneous production of neutrophil extracellular traps during incubation (4 h). The significant NET increase was observed after stimulation with lipopolysaccharides up to 31.59 ± 2.32% after 2-hour incubation period, and up to 42.93 ± 3.56% after 4-hour incubation period. Viferon does not have a significant effect on the number of neutrophilic extracellular traps, but significantly increases their size. Kipferon limits the excessive production of neutrophil extracellular traps, reducing the number of these structures, and also significantly reduces their size and changes their morphology. Ergoferon causes not only a rapid increase in the number of neutrophilic extracellular traps, but also significantly changes their morphology. The extremely long DNA fibers that go beyond the scope of view are observed when exposed to Ergoferon. Kipferon stimulates the production of several morphological forms of neutrophilic extracellular traps at once.Conclusion. The development of innate immune responses is mainly maintained by Viferon and Kipferon. At the same time, Kipferon restrains the intensity of the inflammatory reaction and increases the number of active NK cells. The conducted multilevel study allow researchers to identify new properties and mechanisms of action of administered pharmacological products containing interferon-α, which confirm the effective stimulation of individual components of the immune system due to their action.

Dioscorea alata L. Tubers Improve Diabetes through Anti-hyperglycemia, Anti-inflammation, Ameliorate Insulin Resistance and Mitochondrial Dysfunction

BACKGROUND: Dioscorea alata L. tubers (DA) are suspected to prevent diabetes mellitus (DM) because they have a low glycemic index. However, only a few reports about the anti-diabetic effect of DA were reported up to date. This study aims to analyze the effect of DA consumption on several diabetic biomarkers through in vitro, in vivo, and in silico analysis.METHODS: In vitro experiments were conducted by observing the anti-inflammatory activity of DA extract, steroidal saponins (SDA) isolated from DA, and diosgenin in lymphocyte cell cultures. The tumor necrosis factor (TNF)-α and interferon (IFN)-γ percentages were analyzed by flow cytometry. In vivo study involved 24 healthy adolescents that were given a boiled DA 10 hours post-prandial. The blood sugar levels were measured at 0, 30, 60, and 120 min after treatment. Furthermore, the in silico study was carried out by analyzing the active compounds and predicting the biological activity, the target proteins, and interactions of target proteins with compounds contained in DA .RESULTS: DA extract, SDA isolated from DA, and diosgenin at 50 µg/mL significantly reduced pro-inflammatory cytokines TNF-α and IFN-γ in lymphocyte cell culture. The blood glucose levels in the DA group were lower at 30 and 60 min after treatment. Based on the in silico study, the anti-diabetic activity of DA was speculated to be attributed to the mechanisms of anti-hyperglycemia, prevention of mitochondrial dysfunction, anti-inflammation, and treated insulin resistance. Several proteins included in the DM pathway became the protein target of compounds contained in DA.CONCLUSION: DA potentially have an anti-diabetic activity through several mechanisms.KEYWORDS: hyperglycemia, inflammation, insulin resistance, yam

Immunomodulatory role of edible bone collagen peptides on macrophage and lymphocyte cell cultures

ABSTRACT Immunonutrition or modulation of immune capacity through food and supplements has been gaining significant importance. Hydrolysed collagen has long been used as a functional ingredient, showing multiple physiological activities, including enhancement of immune functions. However, how collagen peptides may affect the immune system still needs further research. This study investigates bone collagen peptides (BCP) immunomodulatory activity on human monocytic THP-1 and human Jurkat T lymphocyte cell lines, using cytokine mRNA expressions as biomarkers. In vitro, gastrointestinal digestion and Caco-2 cell absorption allow obtaining digested and absorbed BCP fractions, respectively, which are tested on immune cells. Results show: (1) Immunostimulatory effect on M0 macrophages, but not on M1 macrophages (lipopolysaccharide (LPS)-activated), (2) Significant T lymphocyte proliferation after incubation with absorbed BCP fraction. (3) Significant increase of anti-inflammatory IL-10 cytokine biomarker. These results suggest that BCP could act as an immunonutrient, modulating the immune response and inflammatory processes. Abbreviations: ATCC: American Type Culture Collection BCP: bone collagen peptide DMSO: dimethyl sulphoxide FBS: fetal bovine serum ECM: extracellular matrix GRAS: Generally recognized as safe; IFN: interferon IL: interleukin, LPS: lipopolysaccharide SEM: standard error of the mean TEER: transepithelial electrical resistance

Evaluation of Oxidant-Antioxidant Status of Fluvoxamine on Human Lymphocyte Cell Culture

Depression is one of the prevalent psychiatric diseases in Turkey and the world. Fluvoxamine is a strong selective serotonin reuptake inhibitor (SSRI) that is used as an antidepressant. No study investigating the doses of Fluvoxamine treatment that may lead to a disruption in the oxidant-antioxidant balance on the cellular level in addition to its beneficial effects has been found in the literature. For this purpose, by creating Fluvoxamine application groups at the doses of 7.5, 15, 30 and 60 μM and durations of 24 and 48 hours on human lymphocyte cell cultures, we investigated the total oxidant status (TOS), total antioxidant status (TAS) and oxidative stress index (OSI). According to the results of the study, the TOS levels of all dose groups increased significantly in the 24- and 48-hour applications (p<0.05). While the TAS levels significantly increased in the 24-hour application of the doses of 7.5 and 15 μM and 48-application of the dose of 7.5 μM, they significantly decreased in the 24- and 48-hour applications of the doses of 30 and 60 μM (p<0.05). The OSI level increased significantly in the 24- and 48-hour applications of the doses of 30 and 60 μM. The results of our study demonstrated that, in the 24- and 48-hour applications of the doses of 30 and 60 μM, the antioxidant system could not compensate and the cells exposed to oxidative stress.

Clostridium perfringens phospholipase C impairs innate immune response by inducing integrated stress response and mitochondrial-induced epigenetic modifications

Clostridium perfringens, a rod-shaped, gram-positive, anaerobic, spore-forming bacterium is one of the most widely occurring bacterial pathogens, associated with a spectrum of diseases in humans. A major virulence factor during its infection is the enzyme phospholipase C encoded by the plc gene, known as Clostridium perfringens phospholipase C (CpPLC). The present study was designed to understand the role of CpPLC in inducing survival mechanisms and mitochondrial-induced epigenetic changes in a human lymphocyte cell culture model. Following exposure to CpPLC, a significant generation of mitochondrial reactive oxygen species was observed, which coincided with the changes in the expression of vital components of MAP/ERK/RTK signaling cascade that regulates the downstream cellular functions. These disturbances further led to alterations in the mitochondrial genome and functioning. This was supported by the observed upregulation in the expression of mitochondrial fission genes Drp1, Fis1, and Mff, and mitochondrial fusion genes MFN1, MFN2, and OPA1 following CpPLC exposure. CpPLC exposed cells showed upregulation of OMA1, DELE1, and HRI genes involved in the integrated stress response (ISR), which suggests that it may induce the ISR that provides a pro-survival mechanism to the host cell. CpPLC also initiated immune patho-physiologic mechanisms including mitochondrial-induced epigenetic modifications through a mitochondrial-ROS driven signaling pathway. Interestingly, epigenetic machinery not only play a pivotal role in lymphocyte homeostasis by contributing to cell-fate decisions but thought to be one of the mechanisms by which intracellular pathogens survive within the host cells. Importantly, the impairment of mtDNA repair among the CpPLC exposed cells, induced alterations within mtDNA methylation, and led to the deregulation of MT-CO1, MT-ND6, MT-ATPase 6, and MT-ATPase8 gene expression profiles that are important for mitochondrial bioenergetics and subsequent metabolic pathways. This was further confirmed by the changes in the activity of mitochondrial electron chain complexes (complex I, II, III, IV and V). The altered mtDNA methylation profile was also found to be closely associated with the varied expression of mitomiRs and their targets. CpPLC exposed cells showed up-regulation of miR24 expression and down-regulation of miR34a, miR150, and miR155, while the increased expression of mitomiR target genes i.e. of K-Ras, MYC, EGFR, and NF-kβ was also observed in these cells. Altogether, our findings provide novel insights into the derailment of redox signaling machinery in CpPLC treated lymphocytes and its role in the induction of survival mechanisms and mitochondrial-induced epigenetic modifications.

Sucralose causes non-selective CD4 and CD8 lymphotoxicity via probable regulation of the MAPK8/APTX/EID1 genes: An in vitro/in silico study.

One of the most widely used sweeteners in the world is sucralose. With sweetening power 600 times greater than sucrose, its use grows among those who seek to cut calories. Research shows that when heated, sucralose generates toxic products that attack the organism and interact with DNA. Our objective was to test this sweetener under unheated conditions and at average concentrations of consumption, evaluating parameters of cytotoxicity, genotoxicity, and immunotoxicity. For this purpose, we made use of lymphocyte cultures and the analysis of their CD3+ , CD4+ , and CD8+ subpopulations. In a complementary way, the mechanism of action is proposed here by computational methods. Our results showed that sucralose reduces non-selectively the total lymphocytes due to falls in the levels of the CD4+ , CD8+ , and CD4+ CD8+ subpopulations. We observed an increase in the level of DNA damage and a gradual incidence of structural changes in the lymphocyte chromosomal sets. It was possible to propose that sucralose modulates the gene expression, interfering especially with the MAPK8, APTX, and EID1 genes. This article presents the results of an evidence-based approach to the safety of human health in the use of sucralose. Finally, this study points out that sucralose has cytotoxic, genotoxic, and mutagenic effects in the concentrations and conditions tested in human lymphocyte cell culture.

Antioxidant effects of dexmedetomidine against hydrogen peroxide-induced DNA damage in vitro by alkaline Comet assay

Background/aimDexmedetomidine (DEX) is an alpha-2 adrenergic agonist that is commonly used as a sedative and anesthetic. The protective effects of DEX against oxidative damage under both in vitro and in vivo conditions have been demonstrated. It was aimed to evaluate and compare the protective effects of DEX and vitamin C (Vit C) on DNA against H2O2-induced DNA damage in human lymphocyte cell cultures in vitro by alkaline Comet assay.Materials and methodsLymphocyte cell cultures were divided into 5 groups, as the negative control, solvent control, positive control, hydrogen peroxide (H2O2; 150 μM) + DEX (1 μM; 2.5 μM; 5 μM), and H2O2 (150 μM) + Vit C (1 μM; 2.5 μM; 5 μM), and incubated at 37 °C for 1 h. Cell viability was measured using the Trypan blue test. DNA damage was measured using the Alkali Comet Technique and the % percent tail intensity was evaluated. Statistical analysis was performed using 1-way ANOVA and the Tukey multiple comparison test.ResultsIt was observed that H2O2 significantly induced DNA damage in the lymphocytes and this damage was decreased significantly with Vit C and DEX. It was observed that Vit C at doses of 1 μM and 2.5 μM had a significantly stronger antioxidant effect, but there was no significant difference between the antioxidant effects of Vit C and DEX with a dose of 5 μM. The dose of 5 μM DEX was found to be the most effective in reducing oxidative DNA damage.ConclusionThere is limited data on the protective effects of DEX against oxidative DNA damage. The primary effect might be cytoprotection. The results herein showed that DEX was protective against H2O2-induced in vitro oxidative DNA damage in lymphocyte cell cultures in a dose-dependent manner. DEX might have a potential therapeutic value in the prevention of oxidative DNA damage in patients.

Vδ1+ T Cells: Adoptive Cell Therapy for the Treatment of Haematological Malignancies in Allogeneic Settings

The treatment of haematological malignancies with adoptive cell therapy such as leukaemia is limited to conventional ab T cells transduced at great cost with chimeric antigen receptors (CAR) recognising the B-cell associated antigen CD19. Although greatly successful especially in paediatric cases, this therapy remains with its challenges such as associated toxicities, relapse rate as well as high production costs and production failure rates due to its autologous nature, indicated by the high risk of αβ T cells inducing sever graft versus host disease (GvHD). In contrast to αβ T cells, γδ T cells are not restricted to, or recognise major histocompatibility complex (MHC) peptide complexes. Especially Vδ1 T cells, enriched in human tissues but also found in small numbers in the human blood have been shown to elicit potent anti-tumour responses recognising stress ligands up-regulated on malignant cells, whilst sparing healthy tissue. A study looking at 39 malignancies, solid tumours as well as haematological malignancies, identified the presence of γδ T cells as the single most positive correlative factor for overall survival. Even more striking, expanding γδ T cells and more specifically Vδ1 T cells correlate with strong graft versus leukaemia and reduced incident rates of GvHD after αβ T cell depleted, haploidentical stem cell transplantation. These correlative findings have been confirmed in several retrospective data-sets but more recently in a prospective study looking at multiple haematological indications such as AML, ALL and MM. We have developed a 2½ week, good manufacturing practice (GMP) compliant and scalable (≥ 1x109) process to consistently generate αβ T cell depleted, γδ T cell lymphocyte cultures (93.5% ± 3.7 of live), strongly enriched for Vδ1 T cells (67.9%±2.2% of live). Vδ1 T cells show good recoverability and proliferation in response to cytokines after cryopreservation, kill a variety of malignant leukaemia and myeloma cells at low effector to target ratios (2:1) and show a favourable innate phenotype expressing high levels of the co-stimulatory molecule CD27, CD69 and the natural cytotoxicity receptors NKG2D, DNAM-1 and NKp30. Expression of negative modulators such as PD-1, CTLA4 and TIGIT remain low or non-detectable. Vδ1 T cells produce large levels of IFN-γ, TNF-α but little to no IL-17 on activation. Furthermore, large amounts of the pro-inflammatory chemokines CCL2 and CLL5, as well as the antigen-presenting cell attracting chemokine CCL4 additionally to the colony stimulating factors M-CSF and GM-CSF, support the idea of Vδ1 T cells orchestrating secondary adaptive immune responses after activation. Importantly, and in accordance with their MHC-independent biology, Vδ1 T cells do not kill healthy lymphocytes or primary tissue-derived stromal cells such as fibroblasts. Vδ1 T cells derived from healthy blood and generated under GMP conditions constitute a potential 'off-the-shelf', fully allogeneic cell therapy for the treatment of multiple haematological malignancies. Disclosures Simoes: GammaDelta Therapeutics Ltd.: Employment. Kawalkowska:GammaDelta Therapeutics Ltd.: Employment. Recaldin:GammaDelta Therapeutics Ltd.: Employment. Foxler:GammaDelta Therapeutics Ltd.: Employment. Nussbaumer:GammaDelta Therapeutics Ltd.: Employment, Equity Ownership, Patents &amp; Royalties. Koslowski:GammaDelta Therapeutics Ltd.: Employment, Equity Ownership.

In Vitro Biocompatibility of Preheated Giomer and Microfilled-Hybrid Composite.

ObjectiveThe aim of this study was to evaluate cytotoxic potencies of two light cured composite materials after heating on different temperatures and cured directly and through CAD/CAM overlay.Materials and methodsComposite materials (microfilled-hybrid Gradia Direct Posterior and Beautifil II) were heated in a Calset warming unit at three different temperatures (T1:37°C, T2:54°C, T3:68°C). A small amount of heated composite material was placed in a round mold (diameter 6mm; 0.65mm thick), covered with Mylar sheet, pressed and polymerized with Bluephase LED unit. One group of samples were polymerized directly, and the other group through 2mm thick CAD/CAM ceramic-reinforced polymer (CRP) and CAD/CAM lithium disilicate ceramic (LDC) overlay for 20 and 40 seconds. The polymerized samples were placed immediately after curing in a lymphocyte cell culture. The viability of peripheral blood lymphocytes was evaluated using a dye exclusion technique by simultaneous staining with ethidium bromide and acridine orange. Quantitative assessments were made by determination of the percentage of viable, apoptotic and necrotic cells. The Pearson chi-square test was used for statistical analysis.ResultsIn case of 20 seconds polymerization, the highest number of viable cells polymerization were recorded when materials were heated at 37°C (T1), while in case of 40 seconds polymerization, the highest number of viable cells were recorded when the materials were heated at 54°C (T2). The samples polymerized through CAD/CAM overlays showed less cytotoxicity than samples polymerized directly.ConclusionApart from composite material composition, the cell viability was also influenced by curing time, temperature of pre-heating and polymerization pattern.

Novel V1551L Mutation in SAMD9L Inhibits Cell Cycle Progression and Results in Pancytopenia That Progresses to MDS with Monosomy 7

Germline SAMD9L mutation is a recently recognized cause of constitutional bone marrow failure with a unique propensity for clonal evolution to myelodysplastic syndrome (MDS) with monosomy 7. It is now known that a clinical syndrome originally described in the 1970s as Ataxia-Pancytopenia Syndrome is caused by missense mutation in SAMD9L, and subsequent case series have demonstrated diverse clinical outcomes in patients with germline SAMD9L mutations. SAMD9L is located on chromosome 7, and gain-of-function mutations in SAMD9L is thought to decrease cell proliferation by inhibiting the normal cell cycle. In patients with SAMD9L mutations, the development of a monosomy 7 clone then rescues the inhibitory effect of mutant SAMD9L but can result in progression to acute leukemia. Interestingly, there are also reports of transient monosomy 7 due to uniparental disomy occurring in the monosomy 7 clone resulting in normal hematopoiesis.We describe the case of a male patient who presented at age 8-months of age with fever, hypotension, and pancytopenia. Bone marrow biopsy was hypocellular with debris-laden histiocytes. Diagnostic evaluation included normal chromosome stability testing (DEB and MMC), normal telomere length testing, and normal gene panels for inherited bone marrow failure and primary HLH. Patient, then, had spontaneous count recovery on day 41 of illness with progressive improvement becoming transfusion independent without any further infections. Repeat bone marrow biopsy 1 month after count recovery demonstrated dysplasia and cytogenetic evaluation revealed monosomy 7 in 11 out of 20 metaphases. Whole exome sequencing demonstrated a novel mutation in SAMD9L. Parents were analyzed for the SAMD9L mutation which were negative. Repeat bone marrow evaluation at 4, 7, and 10 months after presentation continued to demonstrate dysplasia and monosomy 7. After 12 months of observation, decision was made to perform a bone marrow transplant but the patient died from diffuse alveolar hemorrhage shortly after engraftment.EdU incorporation assay showed that 293T cells expressing SAMD9L V1551L mutation exhibit a cell cycle arrest phenotype as shown by the lack of cells in S phase compared to cells expressing the normal SAMD9L protein or empty vector. Furthermore, Digital droplet PCR (ddPCR) with wild type- and mutation-specific primers was used to quantify the percent of mutant allele in the patients' fibroblasts, whole blood, granulocytes, lymphocytes and lymphocyte cell cultures over 12 weeks. The patient's whole blood DNA had a low fraction of the mutation (26.8%), supporting the cytogenetic finding of mosaic monosomy 7. Interestingly, lymphocyte cell line generated from the patient's blood showed a higher fraction of mutation in about 40-45% similar to what is seen in fibroblasts which suggests that the patient's monosomy 7 clone which rescued the neutrophil count seems to preferentially contribute to the myeloid lineage versus the lymphocyte lineage. We have further developed induced pluripotent stem cells from this patient to further explore this question.In summary, our case describes a novel SAMD9L heterozygous missense mutation (p.Val1551Leu) resulting in gain-of-function mutation which inhibits cellular proliferation resulting in bone marrow failure. This case highlights the new insights offered through the use of whole exome sequencing and also the difficulty of making the clinical decision to treat with bone marrow transplant in the setting of multiple reports of disease remission via self-correction. If we are able to identify the clone giving rise to mature cells and quantify it over time, we may be able to provide a more informed recommendation regarding bone marrow transplant versus observation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Toxicology of Pre-heated Composites Polymerized Directly and Through CAD/CAM Overlay.

ObjectivesThe aim was to compare cytotoxicity/genotoxicity of pre-heated composites polymerized through CAD/CAM overlays on isolated human peripheral blood lymphocytes.Material and MethodsA microhybrid (Z100, 3M ESPE) and nanofilled composite (Filtek Supreme Ultra, 3M ESPE) were heated in a heating unit (Calset, AdDent Inc.) at different temperatures: 37 oC, 54 oC, and 68 oC. A small amount of heated composite was placed in a cylindrical mold (6mm diameter; 0.65mm thick), covered with a Mylar sheet, pressed and light-cured directly and through 2 mm thick CAD/CAM ceramic-reinforced polymer (CRP)(LAVA Ultimate, 3M ESPE) or CAD/CAM lithium disilicate ceramic (LDC)(e.max, Ivoclar/Vivadent) overlay. After curing, the specimens were immediately placed in a prepared lymphocyte cell culture. Cytotoxicity was assessed using a dye exclusion method by simultaneous staining with ethidium bromide and acridine orange, aimed to determine percentages of viable, apoptotic and necrotic cells. Genotoxicity was studied using alkaline comet assay.ResultsFor Z100, the highest percentage of viable cells is recorded at T1 (93.7%) after direct light curing, followed by light curing through CRP (92.3%) and through LDC (91.7%T1,T3). For Filtek Supreme Ultra, the highest percentage of viable cells is recorded while curing through CRP (91.0% T2), followed by LDC (90% T1,T3) and direct light curing (88.7%T2).ConclusionFor both tested materials, preheating the procedure at T1 and T2 may be the procedure of choice. In terms of genotoxicity, preheating at T3 may not be suggested.

Cytogenetic tests reveal no toxicity in lymphocytes of rabbit (Oryctolagus cuniculus, 2n=44) feed in presence of verbascoside and/or lycopene

Phenylpropanoid glycosides (PPG), like other phenolic compounds, are a powerful antioxidants and the Verbascoside (VB) is one of the most active of them. A previous study, by using in vitro exposure of blood human lymphocytes to Verbascoside, reported a significant increasings of chromosome fragility compared to control. In the present study, four homogeneous groups of rabbits were used to test in vivo the VB and/or Lycopene (LP) by feeding the animals without VB and LP (control), in presence of VB or/and LP for 80 days. Lymphocyte cell cultures were performed in three different times: 0, 40 and 80 days of the experiment and the cytogenetic tests that we used [CA-test (Chromosome Abnormalities in terms of chromosome and chromatid breaks) and Sister Chromatid Exchange (SCE-test)] have revealed no mutagenic effects on chromosomes. Indeed, mean values/cell of CA and SCE decreased during the experiment with some difference among and within groups, with significant decreasing value only for some group. The study shows clear evidence that diets rich in Verbascoside (and/or Lycopene) do not originate any mutagenic activity, resulting no cytotoxic for the animals and, suggesting a possible their use in both animal and human diets.

TP53 polymorphism in plasma cell myeloma.

Significant and accessible predictive factors for bortezomib treatment in plasma cell myeloma (PCM) are still lacking. TP53 codon 72 polymorphism (P72R) results in proline (P) or arginine (R) at 72 amino acid position, which causes synthesis of proteins with distinct functions. The aims of our study were to: 1) analyze whether this polymorphism is associated with an increased risk of PCM; 2) study whether the P72R polymorphism affects overall survival (OS) among PCM patients; 3) assess the possible association of the P72R polymorphism with sensitivity to bortezomib in cell cultures derived from PCM patients. Genomic DNA from newly diagnosed 59 patients (without IgVH gene rearrangements and TP53 deletions) and 50 healthy blood donors were analyzed by RFLP-PCR to identify TP53 polymorphism. Chromosomal aberrations were detected by use of cIg-FISH. The lymphocyte cell cultures from a subgroup of 40 PCM patients were treated with bortezomib (1, 2 and 4 nM). The P allele of the P72R polymorphism was more common than the R allele in PMC patients compared to controls (39% vs. 24%), and the difference was significant (p = 0.02). The PP and PR genotypes (in combina-tion) were more frequent among cases than in controls (65% vs. 42%, OR = 2.32, p = 0.04). At the cell culture level and 2 nM bortezomib concentration the PP genotype was associated with higher necrosis rates (10.5%) compared to the PR genotype (5.7%, p = 0.006) or the RR genotype (6.3%, p = 0.02); however, no effect of genotypes was observed at bortezomib concentrations of 1 and 4 nM. The shortest OS (12 months) was observed in patients with the PP genotype compared to patients with the PR or RR genotypes (20 months) (p = 0.04). The results suggest that P72R polymorphisms may be associated with an increased PCM risk and may affect OS of PCM patients. However, we saw no consistent results of the polymorphism effect on apoptosis and necrosis in cell cultures derived from PCM patients. Further studies are need in this regard.

Evaluation of Immunotoxicity and Oxidative Stress due to in vitro Exposure of Dimethoate in Chicken Lymphocytes

Pesticide hazards have been amplified by the sharp rise in their agricultural, industrial and domestic use. Though there is considerable contribution of pesticides in various agriculture, domestic and industrial applications; however, continuous exposure of pesticides even at low dose levels can exert adverse effects on mammalian immune system. Dimethoate is an insecticide and acaricide of moderate mammalian toxicity which is used in housefly control and against a broad range of agricultural insect and mite pests. Present communication reports dimethoate induced immunotoxic effects due to enhanced oxidative stress and down regulation of interleukin (IL) levels in avian lymphocyte cell culture system employing lymphocyte proliferation assay, cytokine assay and oxidative stress assay. There was a marked reduction in B and T cells proliferation in avian lymphocytes due to the exposure of thousand times dilution of No Observable Effect Level (NOEL/103) dose of dimethoate. Cytokine assay revealed significant reduction in IL-1 and IL-2 levels. There was a significant increase in oxidative stress in dimethoate treated cells in comparison with controls cells as revealed by nitric oxide estimation. Thus, from our findings, it is revealed that in vitro exposure of low dose of dimethoate induces significant immunotoxic effects and higher oxidative stress in avian lymphocytes.

In vitro Cytotoxic and Genotoxic Evaluation of Morinda tinctoria Roxb. Leaf Extracts

The in vitro cytotoxic and genotoxic evaluation of hexane, chloroform, ethyl acetate, and methanol extracts of Morinda tinctoria leaves was carried out by MTT assay and chromosomal aberration (CA), sister chromatid exchange (SCE), cell proliferation kinetics (CPK), and mitotic index (MI) tests. Five cancer cell lines [SW480 (colon adenocarcinoma); MCF-7 (breast cancer); K562 (chronic myeloid leukemia); NCIH-23 (lung cancer); SKMEL-28 (skin melanoma)] were chosen for the MTT assay. The genotoxic evaluation was carried out on human lymphocyte cell cultures at various concentrations (5, 10, 15, 20 and 25 mg/mL). The results obtained showed (i) concentration-dependent percentage inhibition with the IC50 values of different solvent extracts for cell lines studied ranging from 3.99 to 20.84 mg/mL, (ii) decrease in MI and very slight modification in the CPK values, and (iii) statistical increase in the percentage of cells with CA and the frequency of SCE for cultures exposed to various crude extracts. This is the first report on cytotoxic and genotoxic effects of M. tinctoria leaves extracts. The results suggest that the plant extracts exhibit cytotoxic and possible genotoxic effects with increasing concentration.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT.

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described. In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-γ-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-γ enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented. The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

PRESENT KNOWLEDGE AND EXPERIENCE ON THE STRATEGIES EMPLOYED BY MYCOPLASMA CONTAMINATION OF THE HUMAN CELL CULTURES

Introduction: Mycoplasma species often contaminate cell cultures and other cell-derived biological substances, leading to detrimental effects on the host that include changes in growth, morphology, metabolism and protein synthesis. In cell cultures, mycoplasma are extracellular parasites, usually attached to the external surface of a cell membrane. Many researchers use a mixture of penicillin and streptomycin in the cell culture to prevent contamination. Material and methods: We prepared cell cultures of lymphocytes from peripheral blood of 12 subjects and used micronucleus, assay which is the standard method, for detection of micronuclei in binuclear lymphocytes.Results: Use of standard antibiotics does not protect cell cultures against mycoplasma contamination. Penicillin has no effect on mycoplasma since mycoplasma lack cell wall. Streptomycin inhibits about half the mycoplasma strains but is ineffective against others. In fact, mycoplasma is generally resistant to most antibiotic mixtures commonly used in cell culture. We didn’t find any mycoplasma contamination in the cell culture where penicillin-streptomycin mixture was absent, but confirmed infection in the culture containing mixture of antibiotics.Conclusion: Antibiotics and mixture of antibiotics like penicillin-streptomycin mixture does not protect the cell culture against mycoplasma contamination. Hence, contamination can spread rapidly to other cell lines through aerosol droplet dispersion.

Establishment and characterization of a highly immunogenic human renal carcinoma cell line.

Renal cell carcinoma (RCC) is the most common kidney cancer, and accounts for ~3% of all adult malignancies. RCC has proven refractory to conventional treatment modalities but appears to be the only histological form that shows any consistent response to immunotherapeutic approaches. The development of a clinically effective vaccine remains a major strategic target for devising active specific immunotherapy in RCC. We aimed to identify a highly immunogenic antigenic format for immunotherapeutic approaches, so as to boost immune responses in RCC patients. We established and cloned an immunogenic cell line, RCC85#21 named Elthem, which was derived from a non-aggressive and non-metastatic clear cell carcinoma. The cell line characterization was performed by genomics (real-time PCR, genome instability), proteomics (two dimensional electrophoresis, mass spectrometry) and immunological analysis (mixed lymphocytes tumor cell cultures). Real-time PCR confirmed the RCC85#21 cell expression of tumor antigens and cytokine genes. No difference in microsatellite instability (MSI) in RCC85#21 cell line was found as compared to control, loss of heterozygosity was observed in the RCC85#21 clone, but not in the renal cancer cell lines from which it was generated. The image analysis of RCC85#21 by two-dimensional gels showed 700±26 spots and 119 spots were identified by mass spectrometry analysis. RCC85#21 promoted a significant RCC-specific T cells activation by exhibiting a cytotoxic phenotype after mixed lymphocyte and tumor cell cultures. CD8+ T cells isolated from RCC patients displayed an elevated reactivity against RCC85#21 and efficiently lysed the RCC85#21 clone. The RCC85#21 immunogenic cell line will be suitable for immune stimulation. The identification of novel tumor associated antigens will allow the evaluation of the immune response in vitro and, subsequently, in vivo paving the way for new immunotherapeutic strategies in the RCC setting.

The Effect of Ultra Low Concentrations of Some Biologically Active Substances on the Aerobic Respiration

For today it is known, that primary and secondary disorders of the aerobic respiration, which are based on mitochondrial deficiency, lead to a wide spectrum of clinical manifestations and diseases. Therefore, the question about effective correction of various types of energy exchange disorders remains topical. Thus, the aim of our work was the study effect of the complex of biologically active substances (BAS) in ultra low concentrations on the activity of key enzymes of aerobic energy metabolism succinate dehydrogenase (EC 1.3.99.1) (SQR) and mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) (GPD2). The human lymphocytes assays were tested in vitro (22 donors). In negative control lymphocytes, cell culture normal saline solution was added. Normal saline solution with NaN3 was added in positive control lymphocytes cell culture. Experimental cell culture contained NaN3 and BAS. Our investigations had been revealed increase SQR activity in the experimental cell culture as compared with positive control culture throughout the time of experiment (measurements were carried out at 4, 24, 48, 72 h of incubation). The SQR activity of experimental cell culture and negative control lymphocytes cell culture was equal up to 24 h of experiment. It showed neutralization of NaN3 inhibitory effect (during 24 h) due to BAS influence. Activity of base glycerophosphate shunt ferment GPD2 of experimental lymphocyte cell culture was not different from GPD2 index in the negative control, but was lower than GPD2 activity in the positive control. It also indicated neutralization NaN3 inhibitory effect due to BAS influence. So we had found that extremely low concentrations of selected BAS with their complex impact on human lymphocytes in vitro could effectively neutralize the inhibitory effect of NaN3 on processes of aerobic energy metabolism link.

Absence of toxicity and genotoxicity in an extract of Rubus coriifolius.

Rubus coriifolius Focke is a wild plant from the Rosaceae family. It grows in both Guatemala and Mexico. The polar extract of the aerial parts of this plant has antibacterial, anti-inflammatory, and anti-protozoal activities. These properties may explain the traditional use of this plant. In vivo and in vitro assays were used to assess the genotoxic and toxic effects of an ethanol extract of the aerial parts of R. coriifolius. Three groups of rats were orally administered the R. coriifolius extract diluted in ethanol (5%) at doses of 1.89 mg/kg body weight (low dose), 4.72 mg/kg body weight (medium dose), and 9.44 mg/kg body weight (high dose) for 3 weeks. Genotoxic/cytotoxic effects induced by the R. coriifolius ethanol extract were evaluated in vivo by a micronuclei (MN) test in rat's bone marrow cells and in vitro by MN and sister chromatid exchange (SCE) in human lymphocyte cultures. In vivo genotoxicity analyses revealed that the average number of micronucleated polychromatic erythrocytes and the polychromatic erythrocyte/red blood cell ratio at all doses were not significantly different from those of the negative control. In vitro genotoxicity analyses showed that MN, SCE, and proliferative index frequencies in a human lymphocyte cell culture were not significantly different from those of the negative control. These results demonstrate that the ethanol extract of R. coriifolius aerial parts is not toxic or mutagenic (in vitro and in vivo) and does not affect cell proliferation at the concentrations analyzed.