Human Leukocyte Cultures Research Articles

Primary amenorrhoea - cytogenetic study in 40 Indian women

Background Primary amenorrhoea (PA) refers to an ailment when adolescent girls do not attain menarche naturally. It is one of the most common gynaecological disorders specified. Chromosomal abnormalities play a pivotal role in PA. Cytogenetic analysis is an indispensable diagnostic tool to determine the abnormality of the chromosome. In an emerging country like India, cytogenetic analysis is at a nascent stage. There are very few studies on Cytogenetics present in eastern India, including West Bengal. In rural and suburban areas PA sufferers often experience late diagnosis and struggle to access suitable curative management. The aim of the study is to evaluate the various types of chromosomal abnormalities in patients suffering from PA for accurate, better management of the same and further counselling. Methods A total of 40 PA cases were referred by obstetricians and gynaecologists to the Department of Genetics of Nirnayan Health Care, Kolkata. To screen the chromosomal abnormalities, human leukocyte culture was accomplished with their peripheral venous blood followed by G-banding and then karyotyping was executed according to ISCN-2020. Result Out of 40 patients, 29 were normal among which 46,XX was found in 70% cases (n = 28) and 46,XX,9qh + in 2.5% (n = 1). The remaining 11 showed different types of abnormalities. 45,X was found in 10% (n = 4), 46,X,i(X)(q10) in 2.5% (n = 1), 46,X,del(X)(p11.2) in 2.5% (n = 1), 46,X,del(X)(p22.1) in 2.5% (n = 1), 46,X,del(X)(q24) in 2.5% (n = 1), 46,XY in 2.5% (n = 1), mos 45,X[22]/46,Xi(X)(q.10)[8] in 2.5% (n = 1) and mos 45,X[16]/46,XY[14] (2.5%) in 2.5% (n = 1). Conclusion This study indicates the importance of chromosomal study which must be included in early diagnosis of PA. Karyotyping at the appropriate phase of life will not only help in the judicial management of this disorder but will also give young girls a better lifestyle.

Influence of polyamines of bacterial origin on the production of key cytokines in the culture of human mononuclear leukocytes

To date, participation of biogenic polyamines has been studied in details, with respect to regulation of microbial gene expression, interrelations between bacteria, development of their persistence state. Opportunity of their use as markers of human pathological conditions is being actively evaluated. The aim of our study was to assess the effect of bacterial diamines, i.e., cadaverine and putrescine, upon production of key cytokines (IFNγ and IL-4) in the culture of human mononuclear leukocytes. We studied leukocytes of peripheral venous blood obtained from 18 healthy male volunteers (mean age 24.0±0.6 years). The leukocytes were isolated by means of gradient centrifugation using a Ficoll-Verografin mixture. For the cultivation of lymphocytes, a micro-method and plastic round-bottom 96-well plates were used. Concanavalin A at a concentration of 5 μg/ml was used as a T cell mitogen. Polyamines were used at final concentrations of 5, 25, 50, 75 and 100 μM/L. The cultivation was carried out in humidified atmosphere with 5% CO2, at 37 °C for 72 hours. At the end of incubation, the culture medium was collected and frozen for subsequent quantitative enzyme immunoassays of cytokine concentrations (IFNγ and IL-4) (Russia). Viable cells were counted using Goryaev chamber after staining with 0.1% trypan blue solution. Statistical analysis was performed using Student’s t test or Mann–Whitney test. Addition of cadaverine at all concentrations reduced IFNγ production in the culture of mitogen-activated cells. When culturing leukocytes supplied with putrescine (5 to 50 μM/L), a dose-dependent decrease of IFNγ was observed. Upon further increase of putrescine concentrations, the IFNγ production is restored to the values of the control samples. Direct toxic effect of polyamines upon the cells was revealed. Both the diamines, at the doses of 50, 75, and 100 μM/L, caused increase of IL-4 production by the mitogen-activated cells. Such changes can be associated both with direct cytotoxic effect of cadaverine and putrescine, being mediated by changes of some metabolic pathways. In addition, the effects of polyamines upon monocytes present in culture can include their anti-inflammatory state, e.c., an increased IL-4 production. In general, cadaverine and putrescine, produced by microorganisms of various taxonomic groups, regulate the effectiveness of compensatory-adaptive reactions that ensure adaptation of microbial populations to changing or unfavorable environmental conditions.

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.

Cellular Interactions in Autologous Culture of Human Leukocytes: Mitochondrial Dynamics, Cellular Activation and Extracellular Traps

Cellular Interactions in Autologous Culture of Human Leukocytes: Mitochondrial Dynamics, Cellular Activation and Extracellular Traps

Is tartrazine really safe? In silico and ex vivo toxicological studies in human leukocytes: a question of dose.

The use of food colorings has a long-recorded history. Tartrazine (TRZ) is a dye that confers a lemon-yellow color to food and is widely used in the manufacture of numerous food products, as well as in pharmaceuticals and cosmetics. However, few studies have addressed the toxicology of TRZ in human cells or tissues. Considering the frequent consumption of the TRZ dye in food products and the lack of toxicological data, the present study aimed to evaluate the cytotoxicity and genotoxicity of the TRZ dye in human leukocyte cultures and perform theoretical studies to predict its toxicity in silico. Leukocyte cultures were treated with TRZ at concentrations of 5, 17.5, 35, 70, 100, 200, 300, 400, and 500 μg mL-1. All groups were assayed in triplicates. The mutagenicity was evaluated using the micronucleus test, the nuclear division index, and the nuclear division cytotoxicity index, and the chromosomal instability was quantitatively evaluated by band cytogenetics. Genotoxicity was evaluated using the alkaline comet test. Viability was assessed using the Trypan Blue method. Statistical analyses were performed using analysis of variance followed by Tukey's post hoc test, with a p value <0.05 reflecting statistical significance. No mutagenicity or cytotoxicity was found for the dye at the concentrations evaluated. However, DNA damage was induced by TRZ at a concentration of 70 μg mL-1. These results were confirmed by the predictive data from the in silico evaluations. Further studies are required to confirm our data, considering the frequency of the use of TRZ in the diet of the population, including that of children, as well as the exposure to TRZ through drugs, cosmetics, and other non-food products.

Cytogenetic and micronuclei study of human papillomavirus-related oral squamous cell carcinoma.

Background:The association of human papilloma viral infection in oral squamous cell carcinoma is well studied in the Western countries, but its correlation with DNA damage in the form of micronuclei (MN) formation, ceased apoptosis or presence of chromosomal abnormalities has not yet been studied.Aim:The aim of this study is to find any possible correlation between human papillomavirus (HPV) infection and cytogenetic damage in the oral malignant and premalignant population of West Bengal.Settings and Design:A total of 104 malignant and 103 premalignant cases were selected along with 200 controls.Methods:The buccal smear samples were Pap stained for the detection of MN, apoptosis frequency and koilocytes. The buccal swab samples were processed for DNA extraction followed by polymerase chain reaction for the detection of HPV DNA. The peripheral venous blood samples were processed for the detection of any chromosomal abnormality, by the method of human leukocyte culture followed by Giemsa staining.Statistical Analysis Used:Correlation analysis using GraphPad Prism software was used in this study.Results:About 34.6%, 42.3% and 6.73% of malignant and 6.79%, 3.88% and 20.38% of premalignant cases showed the presence of HPV DNA, koilocytes and apoptosis, respectively. The difference between the MN frequencies of premalignant and malignant oral lesions with the control group is significant with respect to various risk factors (P < 0.05). One percentage of malignant cases showed the presence of chromosomal break.Conclusion:A considerable percentage of malignant cases showing the presence of koilocytes and viral DNA may indicate the effect of HPV infection leading to the malignancy, which can be correlated with the MN and apoptosis frequency.

Antigenotoxic, antimutagenic and cytoprotective potential of Salvia hispanica L. seed extract on human leukocytes exposed to oxidative damage

This study assesses the antigenotoxic activity, antimutagenic and inhibiting cytotoxic conferred for the extract of seeds of Salvia hispanica L. (ShE) in culture of human leukocytes. Phytochemical methods were used by HPLC for determination of compounds: chlorogenic acid, caffeic acid, gallic acid, kaempferol, quercetin and rutin. Cellular cultures were placed in contact with the extract at concentrations of 100µg/mL and 10µg/mL in addition to hydrogen peroxide as causative agent of damage. The results showed a reversal rate of DNA damage of 31%, 41.4% in the frequency of micronuclei, 21.7% in cytoplasmic rates of chromosomal abnormalities and a 17.7% improvement in cell viability in the sample containing 100µg/mL of ShE. The samples that contained the ShE in low concentration of 10µg/mL did not show such reversals against the effects of H2O2. We demonstrated that the use of ShE has beneficial and promising effects.

Evaluation of genotoxic and cytotoxic effects of hydroalcoholic extract of Euphorbia tirucalli (Euphorbiaceae) in cell cultures of human leukocytes.

Euphorbia tirucalli (L.), commonly known as aveloz, has been indiscriminately used in popular medicine to treat various illnesses. However, some components can have devastating consequences. Injury to a cell's genetic material can cause mutations, cancer, and cell death. Our main goal in this work was to evaluate the genotoxic and cytotoxic effects of E. tirucalli extract on human leukocytes. For this purpose, we performed a phytochemical analysis to evaluate the plant's components. In the second step, we treated cultured human leukocytes with different concentrations of the dry extract of the plant and then evaluated the oxidative and genotoxic profiles of these leukocytes. We found that at 1% and 10% concentrations, the aveloz extract acted as a genotoxic agent that could damage DNA and increase oxidative damage. We conclude that despite its popular use, aveloz can act as a genotoxic agent, especially when it contains phorbol ester. Aveloz's indiscriminate use might actually promote tumors and therefore carry a considerable genetic risk for its users.

A comparative study of phytohaemagglutinin and extract of Phaseolus vulgaris seeds by characterization and cytogenetics

Phytohaemagglutinin (PHA) is a lectin obtained from Phaseolus vulgaris (red kidney beans), that acts as a mitogen in human leucocyte culture and is commercially available from Gibco®. This PHA (Gibco®) was found to be very expensive, hence other inexpensive sources that can be used in all kinds of cytogenetics labs (rich and poor), were attempted. One such successful attempt was PHA extract from seeds of P.vulgaris. This paper details the methodology of extraction and application of PHA from seeds of P.vulgaris. Attempts has been made to identify the chemical and physical properties of the products in the extract, analyzed by various spectroscopic and analytical techniques. The analysis clearly indicates that the product from Phaseolus seeds extract was found to be similar to the commercially available PHA (Gibco®) in the cytogenetic study of human leucocyte cultures. The present study enforces the possible utility of the plant extract directly for human leucocyte cultures.

Nipah Virus Uses Leukocytes for Efficient Dissemination within a Host

Nipah virus (NiV) is a recently emerged zoonotic paramyxovirus whose natural reservoirs are several species of Pteropus fruit bats. NiV provokes a widespread vasculitis often associated with severe encephalitis, with up to 75% mortality in humans. We have analyzed the pathogenesis of NiV infection, using human leukocyte cultures and the hamster animal model, which closely reproduces human NiV infection. We report that human lymphocytes and monocytes are not permissive for NiV and a low level of virus replication is detected only in dendritic cells. Interestingly, despite the absence of infection, lymphocytes could efficiently bind NiV and transfer infection to endothelial and Vero cells. This lymphocyte-mediated transinfection was inhibited after proteolytic digestion and neutralization by NiV-specific antibodies, suggesting that cells could transfer infectious virus to other permissive cells without the requirement for NiV internalization. In NiV-infected hamsters, leukocytes captured and carried NiV after intraperitoneal infection without themselves being productively infected. Such NiV-loaded mononuclear leukocytes transfer lethal NiV infection into naïve animals, demonstrating efficient virus transinfection in vivo. Altogether, these results reveal a remarkable capacity of NiV to hijack leukocytes as vehicles to transinfect host cells and spread the virus throughout the organism. This mode of virus transmission represents a rapid and potent method of NiV dissemination, which may contribute to its high pathogenicity.

Improved dendritic cell-based immunization against hepatitis C virus using peptide inhibitors of interleukin 10

The high levels of interleukin 10 (IL-10) present in chronic hepatitis C virus (HCV) infection have been suggested as responsible for the poor antiviral cellular immune responses found in these patients. To overcome the immunosuppressive effect of IL-10 on antigen-presenting cells such as dendritic cells (DCs), we developed peptide inhibitors of IL-10 to restore DC functions and concomitantly induce efficient antiviral immune responses. Two IL-10-binding peptides (p9 and p13) were selected using a phage-displayed library and their capacity to inhibit IL-10 was assessed in a bioassay and in STAT-3 (signal transducer and activator of transcription 3) phosphorylation experiments in vitro. In cultures of human leukocytes where HCV core protein induces the production of IL-10, p13 restored the ability of plasmacytoid DC to produce interferon alpha (IFN-α) after Toll-like receptor 9 (TLR9) stimulation. Similarly, when myeloid DCs were stimulated with CD40L in the presence of HCV core, p9 enhanced IL-12 production by inhibiting HCV core-induced as well as CD40L-induced IL-10. Moreover, in vitro, p13 potentiated the effect of maturation stimuli on human and murine DC, increasing their IL-12 production and stimulatory activity, which resulted in enhanced proliferation and IFN-γ production by responding T-cells. Finally, immunization with p13-treated murine DC induced stronger anti-HCV T-cell responses not only in wildtype mice but also in HCV transgenic mice and in mice transiently expressing HCV core in the liver. These results suggest that IL-10 inhibiting peptides may have important applications to enhance anti-HCV immune responses by restoring the immunostimulatory capabilities of DC.

Anti-Inflammatory Properties of Azelnidipine, a Dihydropyridine-Based Calcium Channel Blocker

Accumulating evidence suggests that inflammation as well as oxidative stress play essential roles in atherogenesis, progression of atherosclerosis, and plaque instability and rupture. Recent studies on available anti-hypertensive agents have focused on their anti-atherosclerotic effects over and above their blood pressure lowering action. These studies have included investigations on several types of calcium channel blockers, with several investigations indicating that a dihydropiridine-based calcium channel blocker, azelnidipine, developed in Japan, has unique anti-oxidative properties. An anti-inflammatory effect of azelnidipine has, however, yet to be established and therefore we carried out a series of in vivo and in vitro studies to investigate this possibility. This was achieved by measuring inflammatory and oxidative stress markers in 16 high risk hypertensive patients administered 16mg/day of azelnidipine. After 4 weeks of treatment, serum levels of hsCRP, IL-6, and IL-8 and urinary 8-OHdG were decreased significantly, despite blood pressure remaining unchanged. Cultures of human mononuclear leukocytes collected from six healthy volunteers showed 100 nM of azelnidipine caused significant inhibition of formyl-methyonyl leucyl phenylalanine (fMLP)-induced production of IL-8. Taken together, these results suggest that azelnidipine has anti-inflammatory effects independent of its anti-hypertensive action. As leukocytes do not possess voltage-operated calcium channels, the effect of azelnidipine in these cells appears to occur independently of an L-type calcium channel antagonizing effect.

Cooperative and Synergistic Activities of the Epithelial Cell Derived Cytokines TSLP, IL-25, and IL-33 on Primary Leukocyte Cultures

RATIONALE: The cytokines thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 individually have been shown to have a profound influence on the development of allergic inflammatory responses. The common epithelial cell origin of these cytokines and overlapping target cell populations suggest that in an appropriate inflammatory state the opportunity for cooperative activities between TSLP, IL-25, and IL-33 may exist. Utilizing a variety of primary human and mouse leukocyte cultures and readouts we examined the individual and combined effects of TSLP, IL-25, and IL-33.

Rosette formation by macrophages with adhered T lymphocytes is precluded by inhibitors of antigen processing and presentation

We had previously found in autologous human leukocyte cultures, in which dead neutrophils phagocytosis by macrophages occur, macrophages and T CD4 lymphocytes perform a selective cell-cell interaction showing many figures of either one, two or several T- lymphocytes adhering to a central macrophage were seen. Considering that antigen presentation would be necessary for the formation of these immune synapses, we attempted to block rosette formation (i.e., the formation of macrophage associations with at least three lymphocytes) by interfering with both antigen processing and presentation. Culture samples of autologous leukocytes from 7 healthy donors were subjected to either brefeldin A, chloroquine or to an anti-HLA DR antibody. Rosette formation was significantly inhibited in the treated samples (either with brefeldin A, chloroquine or the anti- HLA DR; ANOVA, p<0.001, as compared with the untreated controls). It is concluded that interference with antigen processing and presentation precludes the formation of these cell-cell interactions.

Herbicide 2,4‐dichlorophenoxyacetic acid (2,4‐D)‐induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.

Cooperative effects of corticosteroids and catecholamines upon immune deviation of the type-1/type-2 cytokine balance in favor of type-2 expression in human peripheral blood mononuclear cells

A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high levels of catecholamines (CT) and corticosteroids (CS). Although both CS and CT individually can inhibit the production of T-helper 1 (TH1, type-1 like) cytokines and simultaneously promote the production of T-helper 2 (TH2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination CT and CS in immune responses that may be more physiologically relevant. We therefore investigated the combined effects of in vitro CT and CS upon the type-1/type-2 cytokine balance of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of superimposed acute and chronic stress. Results demonstrated a significant decrease in type-1 cytokine production (IFN-γ) and a significant increase in type-2 cytokine production (IL-4, IL-10) in our CS + CT incubated cultures when compared to either CT or CS agents alone. Furthermore, variable enhancement of type-1/type-2 immune deviation occurred depending upon when the CT was added. The data suggest that CS can increase the sensitivity of PBMC to the immunomodulatory effects of CT and establishes an in vitro model to study the combined effects of in vivo type-1/type-2 cytokine alterations observed in acute and chronic stress.

Investigation of Cytokine Expression in Human Leukocyte Cultures with Two Immune-Modulatory Homeopathic Preparations

The efficacy of homeopathic medicines for maintaining human health and treating disease has been extensively examined in clinical trials. However, there is a paucity of preclinical evaluations of the effects of homeopathic medicinal preparations on cellular signaling pathways relevant to the applications of these preparations. In this study, the immune-modulatory effects of Phase 6 (for the stimulation of the nonspecific defense system) and Flu Terminator (for influenza and viral diseases) (Be Well Homeopathics Inc. Miami, FL), two homeopathic preparations developed for the purpose, were evaluated in normal human leukocyte cultures in vitro. Both Phase 6 and Flu Terminator stimulated the production of pro-and anti-inflammatory cytokines by human leukocytes, although higher doses often produced a weaker response than lower doses. The carrier solvent (20% ethanol) failed to elicit any cytokine synthesis. The results of the in vitro studies suggested that ultralow concentrations of ingredients in Phase 6 and Flu Terminator were capable of eliciting a human immune response.

Synthesis of Selenolo[2,3‐b]quinoline‐2‐carboxylic Ethyl Esters (III): Cytogenetic Studies on Human Peripheral Blood Leucocyte Cultures, and Antibacterial Studies, and Antifungal Studies of Their Effects.

Synthesis of Selenolo[2,3‐b]quinoline‐2‐carboxylic Ethyl Esters (III): Cytogenetic Studies on Human Peripheral Blood Leucocyte Cultures, and Antibacterial Studies, and Antifungal Studies of Their Effects.

Synthesis of Selenolo(2,3-b)quinoline-2-carboxylic Ethyl Esters: Cytogenetic Studies on Human Peripheral Blood Leucocyte Cultures, and Anti-Bacterial Studies, and Anti-Fungal Studies of Their Effects

The compounds selenolo(2,3-b)quinoline-2-carboxylic ethyl esters were synthesized in good yields by the reaction of 3-(2-chloro-3-quinolyl)acrylic ethyl esters, with the nucleophilic reagent sodium diselenide in ethanol medium under a nitrogen atmosphere. Cytogenetic studies on human blood leucocytes in vitro were evaluated for some of the synthesized compounds. Most of the synthesized compounds were tested for their antibacterial and antifungal activities.

Cytotoxicity and genotoxicity of low doses of mercury chloride and methylmercury chloride on human lymphocytes in vitro

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 microg/l) of HgCl2 and CH3HgCl and incubated at 37 degrees C for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 microg/l alone, and at 1, 10, 100, and 1000 microg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.