Blood Leukocyte Cultures Research Articles

The Effect of Phytochemical Extracts on Cytokine Gene Expression

Background: In the last century, nutritional supplements have shown a wide spectrum of biochemical effects, most notably about immunomodulation and countering inflammation. Objective: This study investigates the impact of phytochemical compounds that are present in different quantities of pomegranate, grape seeds and garlic extracts on the expression of inflammatory (IL1β and IL6) and anti-inflammatory (IL10) genes, the effects of polymorphisms in these genes on this response. Methods: Human peripheral blood leukocyte cultures were treated with pomegranate (1.2% or 2.4%), garlic (0.5% or 1.2%), or grape seed (1.2% or 2.4%) extracts. Gene expression was assessed with real-time polymerase chain reaction (PCR). Polymorphisms of the cytokine genes were analyzed using allele-specific PCR. Results: Pomegranate extract (2.4%) reduced the transcription of IL1β by 16-fold in comparison to control. The expression of IL6 relative to the control after the addition of grape seed extract (1.2%) was reduced by 100-fold. The grape seeds extract (1.2%) showed the effect of increasing transcription for IL10 compared to the control. The level of IL1β transcription in culture with garlic extract depends on the genotype of the cell for -31T>C polymorphism (r = 0.67 p = 0.03). There is correlation between polymorphism -174G>C and level gene expression IL6 (r=-0.66, p = 0.04) after adding grape seeds extract. Conclusion: The phytochemical compounds in pomegranate extracts and grape seed extracts play the role of anti-inflammatory by decreasing the gene expression of IL1β, IL6 and increasing the transcription of IL10.

Type I Interferon Regulates the Survival and Functionality of B Cells in Rainbow Trout.

Interferons (IFNs) orchestrate antiviral responses in jawed vertebrates and can be classified into three types based on different aspects of their genomic organization, structure and receptors through which they signal and function. Generally, type I and type III IFNs include cytokines that directly induce an antiviral response, whereas type II IFNs are well-known for their immunomodulatory role during viral infections. In mammals, type I IFNs have been shown to also regulate many aspects of B cell development and differentiation. Yet, these functions have been only faintly investigated for teleost IFNs. Thus, in the current study, we have examined the effects of a model type I rainbow trout IFN molecule (IFNa) on blood naïve (IgM+IgD+) B cells, comparing them to those exerted by type II IFN (IFNγ). Our results demonstrate that IFNa increases the survival of naïve rainbow trout B cells, in the absence of lymphoproliferative effects, by rescuing them from spontaneous apoptosis. Additionally, IFNa increased the phagocytic capacity of blood IgM+IgD+ B cells and augmented the number of IgM-secreting cells in blood leukocyte cultures. IFNγ, on the other hand, had only minor effects up-regulating IgM secretion, whereas it increased the phagocytic capacity of IgM− cells in the cultures. Finally, given the recent identification of 9 mx genes in rainbow trout, we have also established which of these genes were transcriptionally regulated in blood naïve B cells in response to IFNa. This study points to a previously undescribed role for teleost type I IFNs in the regulation of B cell responses.

Influence of polyphenols from olive mill wastewater on the gastrointestinal tract, alveolar macrophages and blood leukocytes of pigs

In the last years, great importance has been given to the beneficial effects of polyphenols. Among the most relevant health promoting effects, there is the capacity to reduce the amount of free radicals and stimulate the immune response. In this study, polyphenols extracted from olive mill wastewater (OMWW), were fed to adult ‘Casertana’ pigs during the finishing period. No significant differences in the length of the jejunum-ileum villi and the depth of the colon crypts were detected between control and polyphenols fed pigs. Instead, intra-epithelial and lamina propria leukocytes were more abundant in pigs fed polyphenols (p < .05). Cyclooxygenase-2 immunoreactivity in the gastrointestinal tract, employed as marker of inflammation, was more intense in the control group. Superoxide anion production in primary cell cultures of both blood leukocytes and alveolar macrophages was lower in pigs fed polyphenols (p < .05). Taken together these data indicate that, according to our in vitro studies, OMWW polyphenols seem to be potent antioxidants, while the interpretation of the in vivo experiments is more problematic and further studies are necessary on the interactions between bioactive feed compounds and intestinal status. Such studies can contribute to a better understanding of both positive and negative interactions in vivo and to the identification of new functional feeds.HIGHLIGHTSThe effects of polyphenols extracted from olive mill wastewater (OMWW) have been studied in pigs.Gut morphology, inflammation and immune response were investigated.OMWW polyphenols act as potent antioxidants.

The Effect of Three Different Suture Anchors for Rotator Cuff Repair on Primary Cultures of Human Bone Marrow Mesenchymal Stem Cells.

Purpose The purpose of this study is to investigate the in vitro biocompatibility of three different suture anchors (all-suture anchor, metal anchor, and polyetheretherketone anchor), commonly used for the rotator cuff repair. Methods To assess the biocompatibility of the anchors, the possible cytotoxicity and the immunogenicity of the devices were assessed by cell viability assay and cell count on cultures of bone marrow stem cells (BMSCs) and peripheral blood leucocytes (PBLs), respectively. The possible inhibitory effect of the devices on BMSCs osteogenic potential was evaluated by alkaline phosphatase activity and matrix deposition assay. Results The viability of BMSCs was slightly reduced when cultured in the presence of the devices (−24 ± 3%). Nevertheless, they were able to differentiate toward the osteogenic lineage in all culture conditions. The proliferation of PBLs and the production of interleukin-2 were not enhanced by the presence of any device. Conclusion The analyzed devices did not significantly affect the normal cells functions when directly cultured with human primary BMSCs or PBLs, in terms of osteogenic differentiation and inflammatory reaction. Clinical Relevance A deeper knowledge of the biological reactions to different devices used in rotator cuff surgeries would improve the clinical outcome of these procedures.

Blood Cells and Leukocyte Culture - A Short Review

Blood Cells and Leukocyte Culture - A Short Review

Platelet survivability in blood leukocyte culture

Platelet survivability in blood leukocyte culture

Aging in Ghana

Interferon (IFN)-gamma production, stimulated by the addition of exogenous interleukin (IL) 2, T cell mitogens, or tuberculin purified protein derivative (PPD) was studied in cultures of separated human mononuclear cells or unseparated peripheral blood leukocytes (PBL). IFN-gamma was induced by the addition of IL 2 to cultures of otherwise unstimulated cells. The minimal concentration of exogenous IL 2 required to cause a reproducible stimulation of IFN-gamma was about 10 U/ml, i.e., approximately 50 times the minimal concentration required to stimulate proliferation in an IL 2-dependent murine cytotoxic T cell line. Approximately 500 to 1000 IL 2 U/ml were required to produce maximal stimulation of IFN-gamma production in otherwise unstimulated cultures. Monoclonal antibody anti-Tac, specific for an epitope associated with the IL 2 receptor (IL 2 R), inhibited IFN-gamma induction by exogenous IL 2 less strongly than induction by phytohemagglutinin (PHA) or concanavalin A (Con A). The highest degree of inhibition was exerted by anti-Tac on IFN-gamma production stimulated with PPD. Stimulation of IFN-gamma induction by exogenous IL 2 and the inhibitory action of anti-Tac on IFN-gamma production were also seen in cultures of irradiated (2000 R) cells. Treatment of cells with subinducing doses of Con A or phorbol myristate acetate increased IFN-gamma induction by exogenous IL 2. Taken together, the data suggest that endogenously generated IL 2 is a major mediator of IFN-gamma induction in PBL cultures stimulated with antigens or T cell mitogens.

Carica papaya induces in vitro thrombopoietic cytokines secretion by mesenchymal stem cells and haematopoietic cells.

BackgroundUse of Carica papaya leaf extracts, reported to improve thrombocyte counts in dengue patients, demands further analysis on the underlying mechanism of its thrombopoietic cytokines inductionMethodsIn vitro cultures of peripheral blood leukocytes (PBL) and stem cells from human exfoliated deciduous teeth (SHED) were treated with unripe papaya pulp juice (UPJ) to evaluate its potential to induce thrombopoietic cytokines (IL-6 and SCF)ResultsIn vitro scratch gap closure was significantly faster (p < .05) in SHED culture treated with UPJ. IL-6 concentration was significantly increased (p < .05) in SHED and PBL culture supernatant when treated with UPJ. SCF synthesis in SHED culture was also significantly increased (p < .05) when treated with UPJConclusionIn vitro upregulated synthesis of IL −6 and SCF both in PBL and SHED reveals the potential mechanism of unripe papaya to induce thrombopoietic cytokines synthesis in cells of hematopoietic and mesenchymal origin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0749-6) contains supplementary material, which is available to authorized users.

IL-22 and IL-22 binding protein (IL-22BP) regulate fibrosis and cirrhosis in hepatitis C virus and schistosome infections.

Interleukin (IL)-22 acts on epithelia, hepatocytes, and pancreatic cells and stimulates innate immunity, tissue protection, and repair. IL-22 may also cause inflammation and abnormal cell proliferation. The binding of IL-22 to its receptor is competed by IL-22 binding protein (IL-22BP), which may limit the deleterious effects of IL-22. The role of IL-22 and IL-22BP in chronic liver diseases is unknown. We addressed this question in individuals chronically infected with schistosomes or hepatitis C virus (HCV). We first demonstrate that schistosome eggs stimulate production of IL-22 transcripts and inhibit accumulation of IL22-BP transcripts in schistosome-infected mice, and that schistosome eggs selectively stimulate production of IL-22 in cultures of blood leukocytes from individuals chronically infected with Schistosoma japonicum. High IL-22 levels in cultures correlated with protection against hepatic fibrosis and portal hypertension. To test further the implication of IL-22/IL-22BP in hepatic disease, we analyzed common genetic variants of IL22RA2, which encodes IL-22BP, and found that the genotypes, AA, GG of rs6570136 (P = 0.003; odds ratio [OR] = 2), and CC, TT of rs2064501 (P = 0.01; OR = 2), were associated with severe fibrosis in Chinese infected with S. japonicum. We confirmed this result in Sudanese (rs6570136 GG [P = 0.0007; OR = 8.2], rs2064501 TT [P = 0.02; OR = 3.1]), and Brazilians (rs6570136 GG [P = 0.003; OR = 26], rs2064501 TC, TT (P = 0.03; OR = 11]) infected with S. mansoni. The aggravating genotypes were associated with high IL22RA2 transcripts levels. Furthermore, these same variants were also associated with HCV-induced fibrosis and cirrhosis (rs6570136 GG, GA [P = 0.007; OR = 1.7], rs2064501 TT, TC (P = 0.004; OR = 2.4]). These results provide strong evidence that IL-22 protects against and IL-22BP aggravates liver fibrosis and cirrhosis in humans with chronic liver infections. Thus, pharmacological modulation of IL-22 BP may be an effective strategy to limit cirrhosis.

Essential oil from Ageratum fastigiatum reduces expression of the pro-inflammatory cytokine tumor necrosis factor-alpha in peripheral blood leukocytes subjected to in vitro stimulation with phorbol myristate acetate

Ageratum fastigiatum (Gardner) R.M. King & H. Rob., a member of the Asteraceae family popularly known in Brazil as “matapasto”, is indicated in folk medicine as anti-inflammatory and analgesic. Despite its popular use, little is known about its potential effect on the parameters involved in an inflammatory response. The objective of this study was to characterize the chemical composition of the essential oil from A. fastigiatum and to evaluate the frequency of tumor necrosis factor alpha and interferon gamma producing cells in peripheral blood lymphocytes stimulated with phorbol myristate acetate in the presence of essential oil from A. fastigiatum. Non-toxic concentrations of essential oil from A. fastigiatum were evaluated in cultures of peripheral blood leucocytes using the trypan blue exclusion assay by flow cytometry. GC–MS analysis revealed that the prevalent compounds identified in the essential oil from A. fastigiatum sample were α-pinene, limonene, trans-caryophyllene, α-humulene, caryophyllene oxide, 1,2-humulene-epoxide, 1,6-humulanodien-3-ol, and α-cadinol. Results showed that exposure to essential oil from A. fastigiatum at concentrations of 0.5×10−2 and 1×10−2μl/ml caused no alterations in leukocyte viability as compared to the control group. Both concentrations lowered the percentage of tumor necrosis factor alpha (+)-lymphocytes and neutrophils. There were no changes in the percentage of lymphocytes positive for the interferon gamma cytokine. Our results suggest that part of the anti-inflammatory activity attributed to A. fastigiatum may be due to the effect of some of its components in decreasing the number of cells that produce the pro-inflammatory cytokine tumor necrosis factor alpha.

SIMULATION OF PROTECTIVE IMMUNE MECHANISMS AT AFRICAN SWINE FEVER in vitro

S u m m a r y Pigs that survive after infection with African swine fever virus are resistant to reinfection with seroimmunotype-homologous virulent isolates, indicating an immune protection development. Nevertheless, the points regarding the protective importance of various immune mechanisms at African swine fever still remain controversial. Peripheral blood leukocyte cultures prepared from both intact or vaccinated pigs were used as models of implemented virus-specific reactions in vivo. The maximum amount of ASF virus was determined in the cultures with adherent cells from intact or immunized gilt, non-adherent white blood cells and the serum of an intact animal. Replacement of non-adherent cells from intact gilt by those from the immunized one led to a significant decrease in ASF virus titres. It was shown that combination of A-cells from intact or immunized gilt, non-adherent cells from the intact animal and the serum from immunized animal limited the viral propagation more strictly, under the replacement of non-adherent cells from the intact gilt by those from the immunized animal at the most. The results determine the role of cellular and humoral immunity in limiting African swine fever virus replication. The protection mechanisms mediated by cytotoxic T lymphocytes and antibody-mediated cellular anti-effectors are found to have an integrated effect, suggesting their targeting against different epitopes.

МОДЕЛИРОВАНИЕ in vitro ЗАЩИТНЫХ ИММУННЫХ МЕХАНИЗМОВ ПРИ АФРИКАНСКОЙ ЧУМЕ СВИНЕЙ

Pigs that survive after infection with African swine fever virus are resistant to reinfection with seroimmunotype-homologous virulent isolates, indicating an immune protection development. Nevertheless, the points regarding the protective importance of various immune mechanisms at African swine fever still remain controversial. Peripheral blood leukocyte cultures prepared from both intact or vaccinated pigs were used as models of implemented virus-specific reactions in vivo. The maximum amount of ASF virus was determined in the cultures with adherent cells from intact or immunized gilt, non-adherent white blood cells and the serum of an intact animal. Replacement of non-adherent cells from intact gilt by those from the immunized one led to a significant decrease in ASF virus titres. It was shown that combination of A-cells from intact or immunized gilt, non-adherent cells from the intact animal and the serum from immunized animal limited the viral propagation more strictly, under the replacement of non-adherent cells from the intact gilt by those from the immunized animal at the most. The results determine the role of cellular and humoral immunity in limiting African swine fever virus replication. The protection mechanisms mediated by cytotoxic T lymphocytes and antibody-mediated cellular anti-effectors are found to have an integrated effect, suggesting their targeting against different epitopes.

Study among betel quid chewers from Indian population

Background: Oral cancer is most common in males and also in females. Betel quid (BQ) is the main causative agent of oral cancer. Areca catechu, a major component of BQ, contains certain alkaloids that give rise to nitrosamines. Mitotic index (MI) and Micronuclei (MN) were studied among the studied population. Methods: In this present study subjects were screened from Department of E.N.T. & Department of Oral and Facio maxillary of RKMSP hospital, and different areas of Eastern and North Eastern states of India. For mitotic index (MI) blood leukocyte cultures were analyzed and for micronuclei (MN) buccal mucosa were examined. Results: Some of them had more than one addiction. Micronuclei percentage and mitotic index both higher than normal. Conclusion: Betel quid play a role in changing the oral pathology and thus causes oral cancer.

Release of reactive oxygen species and production of nitric oxide in the cultures of peripheral blood leukocytes of young athletes

Release of reactive oxygen species and production of nitric oxide in the cultures of peripheral blood leukocytes of young athletes

Sour Cherry Seed Kernel Extract Increases Heme Oxygenase‐1 Expression and Decreases Representation of CD3+ TNF‐α + and CD3 + IL‐8+ Subpopulations in Peripheral Blood Leukocyte Cultures from Type 2 Diabetes Patients

The present study evaluates a hypothesis that sour cherry (Prunus cerasus) seed extracts (SCE) modulate CD3+ T lymphocyte activity in ways predictive of potential for uses of SCE in management of inflammatory diseases. Peripheral blood mononuclear cells (PBMC) from 12 type 2 diabetes (T2DM) patients and eight healthy control subjects were cultured 24 h with 100 ng/ml lipopolysaccharide (LPS) to increase inflammatory signaling and co-incubated with 0.5-100 µg/ml SCE. Cultures were evaluated by two-color flow cytometry for percent representation of CD3+ IL8+ and CD3+TNF-α cells which express interleukin-8 (IL-8), and tumor necrosis factor-α, (TNF-α+) respectively, and by enzyme-linked immunoassay for lymphocyte-associated heme oxygenase-1 (HO-1, known to be induced by SCE). SCE dosage ranges of 0.5-100 µg/ml in cell cultures significantly suppressed LPS-increased CD3+TNF-α+ and CD3+IL8+ representation from all participants (p < 0.05), with greater pharmacological effect noted in suppression of CD3+TNF-α+ noted in cells from T2DM patients versus healthy control subjects. These effects correlated with increased HO-1 expression in SCE-treated PBMC from all subjects (p < 0.05). Since TNF-α and IL-8 are diagnostic/prognostic biomarkers for many inflammatory syndromes, the capacity of SCE to down-regulate representation of cells that express them suggests potential for therapeutic use of SCE in T2DM and other diseases.

CXCL8 of Scophthalmus maximus: Expression, biological activity and immunoregulatory effect

CXCL8, or interleukin-8, is a CXC chemokine that promotes neutrophil migration in response to inflammatory stimuli. In this study, we identified and analyzed a CXCL8 orthologue, SmCXCL8, from turbot (Scophthalmus maximus). The deduced amino acid sequence of SmCXCL8 is 99-residue in length and shares 52–83% overall identities with the lineage 1 CXCL8 of a number of teleost. SmCXCL8 possesses a CXC chemokine domain that contains the conserved CXC motif preceded by the tripeptide sequence EMH. Purified recombinant SmCXCL8 (rSmCXCL8) induced chemotaxis in peripheral blood neutrophils and, to lesser extents, head kidney (HK) lymphocytes and macrophages in a dose-dependent manner. Mutation of the EMH motif by alanine substitution reduced the chemoattractive effect of rSmCXCL8. Expression of SmCXCL8 as determined by quantitative real time RT-PCR (qRT-PCR) was detected mainly in immune organs under normal physiological conditions and was upregulated by experiment challenges with bacterial pathogen and poly(I:C). In addition, SmCXCL8 expression was also induced to significant extents following vaccination of turbot with a subunit vaccine. When rSmCXCL8 was added to the cell cultures of peripheral blood leukocytes and HK lymphocytes and macrophages, it stimulated the proliferation of these cells and enhanced cellular resistance against intracellular bacterial survival. qRT-PCR analysis showed that rSmCXCL8 induced the expression of TNF-α and suppressor of cytokine signaling 3 in HK lymphocytes in different time frames. On the other hand, SmCXCL8 expression was also upregulated by TNF-α. Taken together, these results indicate that SmCXCL8 is a functional CXC chemokine with immunomodulatory effect and plays a role in inflammatory response induced by bacterial infection.

Genotoxic Effect of Dye Effluents in Chromosomes of Indigenous Goats (Capra hircus)

The genotoxic effect of dye effluents was assessed in indigenous goats inhabiting textile industry areas encompassing dyeing and bleaching units by means of mitotic drive, mitotic index, chromosome aberrations and the mean SCEs frequency. Peripheral blood leucocyte cultures were set up with culture medium and pokeweed mitogen. The cultures were supplemented without bromodeoxyuridine (BrdU) for chromosome aberrations and with BrdU for the mean SCEs frequency for 2 cell cycles. Air-dried slides were stained with Giemsa for chromosome aberrations and the fluorescent plus Giemsa (FPG) technique was adopted for the display of sister chromatid differentiation and the estimation of the mean SCEs frequency. No gross structural and numerical chromosome abnormalities were noticed in all animals screened based on G-banding and karyotyping. The mitotic drive for the control and exposed populations was 37.58±1.29 and 26.13±0.78%, respectively. The mitotic drive in exposed population was significantly (p<0.01) lower when compared to control animals. The mitotic index for the control and exposed populations was 12.49±0.42 and 11.49±0.43%, respectively. Though there was a decrease in mitotic index in the exposed population, it was not statistically significant. The frequency of SCEs did not follow the Poisson distribution in both the control and exposed populations of goats. The pooled mean±S.E (range) of SCEs frequency for the control and exposed populations of goats were 4.83±0.16 (1–11) and 12.98±0.16 (8–19), respectively. There was significant (p<0.01) increase in the mean SCEs frequency of goats (exposed) reared in industrial areas. The number of SCEs in chromosome 1 was significantly (p<0.05) higher than that expected from relative chromosomal length.

Tropical Chronic Pancreatitis (TCP): A Cytogenetic Study in Patients Residing in and around Coimbatore City, Tamil Nadu

KEYWORDS Tropical. Pancreatitis. Karyotype. Chromosomal Aberrations ABSTACT Tropical chronic pancreatitis (TCP) is a juvenile form of chronic calcific non- alcoholic pancreatitis found almost exclusively in tropical countries. This study was performed to find out the chromosomal aberrations in chronic pancreatitic patients of Coimbatore city, south India. 41 tropical chronic pancreatitis patients were examined for chromosome abnormalities using standard procedure of analysis from peripheral blood leukocyte culture using trypsin G – banding. The patients and the control set of individuals displayed different types of chromosomal abnormalities such as deletions, translocations, inversions and mosaicism. Familial transmission of chromosomal aberration was detected in 6 TCP patients and abnormalities in chromosome 7 were found in seven patients including the familial cases. The etiology of the disease needs to be studied, taking geographical location of Coimbatore city also into consideration.

Chronic Intestinal Helminth Infections Are Associated with Immune Hyporesponsiveness and Induction of a Regulatory Network

Helminth infections have been associated with protection against allergy and autoimmune diseases. We investigated the effects of chronic infections with Ascaris lumbricoides and Trichuris trichiura (measured twice over a 5-year period) on cytokine and antibody responses. We collected blood from 1,060 children aged 4 to 11 years living in a poor urban area of Brazil and measured Th1 (gamma interferon [IFN-gamma]) and Th2 (interleukin-5 [IL-5] and IL-13) cytokines and the regulatory cytokine IL-10 in unstimulated and stimulated (with mitogen or A. lumbricoides antigens) cultures of peripheral blood leukocytes and levels of total IgE and anti-A. lumbricoides IgG4 and IgE in serum. Intestinal helminth infections were associated with an increased proportion of children producing IL-5 in response to A. lumbricoides and producing IL-10 spontaneously, especially among coinfected and chronically infected children. Helminth infections were associated with a generalized suppression of cytokine responses to mitogen. Levels of total IgE and anti-A. lumbricoides IgG4 and IgE were especially elevated in chronically infected children. In conclusion, intestinal helminth infections were associated with a typical Th2 immune response profile and with the induction of immune hyporesponsiveness that was associated with greater frequencies of the production of spontaneous IL-10.

Tropical Chronic Pancreatitis (TCP): A Cytogenetic Study in Patients Residing in and around Coimbatore City, Tamil Nadu

KEYWORDS Tropical. Pancreatitis. Karyotype. Chromosomal Aberrations ABSTACT Tropical chronic pancreatitis (TCP) is a juvenile form of chronic calcific non- alcoholic pancreatitis found almost exclusively in tropical countries. This study was performed to find out the chromosomal aberrations in chronic pancreatitic patients of Coimbatore city, south India. 41 tropical chronic pancreatitis patients were examined for chromosome abnormalities using standard procedure of analysis from peripheral blood leukocyte culture using trypsin G – banding. The patients and the control set of individuals displayed different types of chromosomal abnormalities such as deletions, translocations, inversions and mosaicism. Familial transmission of chromosomal aberration was detected in 6 TCP patients and abnormalities in chromosome 7 were found in seven patients including the familial cases. The etiology of the disease needs to be studied, taking geographical location of Coimbatore city also into consideration.