Single-wall carbon nanotubes (SWCNT) induce cytotoxicity and genotoxicity produced by reactive oxygen species (ROS) generation in phytohemagglutinin (PHA)-stimulated male human peripheral blood lymphocytes.

Single-wall carbon nanotubes (SWCNT) possess a small size, large surface area, and high reactivity, which enable them to permeate the cytoplasmic or nuclear membrane and attach to biological molecules. During medical applications, SWNCT are usually administered intravenously, which enhances interaction with blood components. Yet despite this exposure potential, safety evaluation studies of SWCNTs focused on human blood cells are still lacking. Therefore, this study was undertaken to examine cytotoxicity, genotoxicity, and proinflammatory responses following SWCNT treatment of phytohemagglutinin (PHA)-stimulated male human peripheral blood lymphocytes (PBL). SWCNT were found to inhibit cell growth, as well as to induce DNA breakage, and micronuclei (MN) formation via reactive oxygen species (ROS) generation. The addition of N-acetylcysteine (NAC) a cell-permeable antioxidant, decreased ROS generation, cytotoxicity, and genotoxicity produced by SWCNT treatment. In addition, SWCNT induced tumor necrosis factor (TNF)-α release after 24 h, yet this phenomenon was not related to ROS generation, as antioxidant NAC treatment did not affect increased proinflammatory cytokine levels in the phytohemagglutinin (PHA)-stimulated male human PBL.

Current status of the acquired immune system of Iranian patients with long-term complications of sulfur mustard poisoning.

BACKGROUND: Sulfur mustard (SM) is a powerful blistering chemical warfare agent that has genotoxic effects. Cells with excessive proliferation such as lymphocytes may inherit this cellular toxicity which can lead to their malfunctions in the long-term. This study was designed to evaluate the status of acquired immunity among SM poisoned veterans around three decades after exposure. METHODS: Thirty five male Iranian veterans having at least 25% disability due to SM poisoning with long-term complications in the respiratory system, skin or eyes were investigated. Non-functional/functional tests including hematological parameters, immunostaining analysis, lymphocyte proliferation assay, cytokine profile, and levels of total serum IgM, IgG and IgA were performed. RESULTS: The results showed that most of the parameters of adaptive immune system of the veterans were currently within the normal ranges. However, changes in the proliferation index (PI) of lymphocytes showed problems with the lymphocytes which cannot be proliferated appropriately. PI values for PBMCs (peripheral blood mononuclear cells) in presence of PHA (Phytohemagglutinin-A) and LPS (lipopolysaccharide) mitogens were 1.16 ± 0.14 and 1.13 ± 0.07, respectively which are less than expected. CONCLUSIONS: Based on the results gathered in this study, most of the parameters of acquired immunity were normal. However, the observed failure of lymphocyte functions may disrupt physiological activity of whole immune system leading to long-term complications; including recurrent respiratory tract infections. Indeed, further cellular and molecular studies with regard to lymphocytes function are required to better understand the status of adaptive immunity in these patients. Graphical abstract ᅟ.

Increased micronucleus frequency in phytohaemagglutinin-stimulated blood cells of patients with vitiligo.

BACKGROUND: Vitiligo is a relatively common, acquired pigmentary disorder characterized by areas of depigmented skin resulting from loss of melanocytes in the epidermis. Although several hypotheses have been proposed for the aetiology and pathogenesis of vitiligo, the cause of vitiligo remains unclear. OBJECTIVE: To evaluate spontaneous micronucleus (MN) frequency using the cytokinesis block MN assay to determine damages at the DNA or chromosome level in phytohaemagglutinin (PHA)-stimulated blood cells of patients with vitiligo and healthy control subjects. METHODS: Peripheral blood samples were obtained and cultured from 21 patients with vitiligo (mean age: 21.48 +/- 9.78 years) and 21 age- and sex-matched healthy control subjects (mean age: 21.52 +/- 9.80 years). MN values were scored in binucleated cells obtained from whole-blood cultures of patients and control subjects. RESULTS: MN frequencies (mean +/- SD) in PHA-stimulated blood cells of patients with vitiligo and control subjects were 0.94 +/- 0.58 and 0.58 +/- 0.32, respectively. Compared with control subjects, MN frequencies of patients with vitiligo were found significantly higher than those of the control subjects (P = 0.012). CONCLUSION: Our results indicate unexpectedly some chromosomal/DNA damage in whole-blood cultures of patients with vitiligo. We do not know, however, if these chromosome/DNA instabilities observed in the cells of vitiligo patients resulted from the cause or from the consequences of the disorder.

Effect of White Kidney Beans (Phaseolus vulgaris L. var. Beldia) on Small Intestine Morphology and Function in Wistar Rats.

The chronic ingestion of raw or undercooked kidney beans (Phaseolus vulgaris L.) causes functional and morphological derangement in various tissues. The major objectives of this study were to investigate the gavage effects of a raw Beldia bean variety that is widely consumed in Tunisia, on the small intestine morphology and jejunal absorption of water, electrolytes, and glucose in Wistar rats. Twenty young male rats were randomly divided into two groups of 10 rats. The first group served as the control and was gavaged with 300 mg of a rodent pellet flour suspension (RPFS), whereas the second experimental group was challenged with 300 mg of a Beldia bean flour suspension (BBFS) for 10 days. Histological studies were performed using light and electron microcopy. The intestinal transport of water, sodium, potassium, and glucose was studied by perfusing the jejunal loops of the small bowels in vivo. The feeding experiments indicated that BBFS did not affect weight gain. Histomorphometric analyses showed that the villus heights, crypt depths, and crypt/villus ratios in the jejunum and ileum were greater in the BBFS-fed rats than controls. Electron microscopy studies demonstrated that the rats exposed to RPFS exhibited intact intestinal tracts; however, the BBFS-treated rats demonstrated intestinal alterations characterized by abnormal microvillus architectures, with short and dense or long and slender features, in addition to the sparse presence of vesicles near the brush border membrane. BBFS administration did not significantly affect glucose absorption. However, significant decreases were observed in water and electrolyte absorption compared with the uptake of the controls. In conclusion, raw Beldia beans distorted jejunum morphology and disturbed hydroelectrolytic flux.

[Lectin-induced platelet aggregation inpatients with unstable angina pectoris treated with anti-aggregation agents]. / Lektiniindutsirovannaia agregatsiia trombotsitov u bol'nykh s nestabil'noi stenokardiei na fone priema antiagregantov.

Lectin-induced platelet aggregation was studied in patients with unstable angina pectoris treated with tiklid and aspirin. ADP, PHA-P, WGA, Con A served as the aggregation inductors. In unstable angina pectoris lectin-induced platelet aggregation was enhanced. Changes in this aggregation in response to treatment with antiaggregants with different mechanisms of action are demonstrated.

[Methods of achieving group-specific activity of plant extracts]. / O metodakh dostizheniia gruppospetsificheskoi aktivnosti rastitel'nykh ékstratov.

Different methods of eliminating the side effects of phytohemagglutinins and of enhancing the titer of a needed phytohemagglutinin are described. The importance of the above methods for the forensic-medicine serology is demonstrated.

Tannins, trypsin inhibitors and lectin cytotoxicity in tepary (Phaseolus acutifolius) and common (Phaseolus vulgaris) beans.

This study compared the levels of antinutritional components and cytotoxic effect of extracts, from tepary (Phaseolus acutifolius) and common (Phaseolus vulgaris) beans. Antinutritional factors were evaluated by determining their effect on the viability of epithelial cells isolated from rat small intestine. The protein and carbohydrates content were similar in all the genotypes studied (20 and 60%, respectively). Common beans presented higher content of trypsin inhibitors, tannins and lectins than tepary beans. There was not a significant correlation between tannins and cooking time. However, water absorption and cooking time correlated significantly (p < 0.05). Considerable variation was observed in lectin activity (1302-18161 Ul/mg) of extracts from different beans. Tannins, lectins, trypsin inhibitors and fat content differed between bean varieties whereas protein content was similar. The percent cellularity on rat epithelial cells was significantly different among protein extracts from different bean cultivars and ranged between 53.5% and 87.4% (p < 0.05). These results suggest that the incorporation of tepary beans in the diet would not alter the current nutritional contribution of common beans or introduce adverse toxic effects. The agronomic characteristics of tepary beans make them attractive for cultivation. However, the harder to cook phenomenon may be a limiting factor that needs further consideration.

Red kidney beans--to eat or not to eat?

The importation of dry red kidney beans (a variety of the species Phaseolus vulgaris) for cultivation or consumption in South Africa is prohibited because of their potential toxicity to humans. It has been established that the haemagglutinating lectins (e.g. phytohaemagglutinin (PHA)) in kidney beans are responsible for this toxicity. Dry bean varieties available on the South African market for human consumption as well as locally produced (for this study) and imported dry red kidney beans and imported canned red kidney beans were compared. The PHA activity and the effect of heat thereon were measured, before and after overnight soaking. The PHA activity in extracts of uncooked and incompletely cooked red kidney beans was not higher than the levels measured in 50% of the other bean varieties included in the study. These findings indicate that the toxic potentials and health risks associated with red kidney beans are similar to those of other dry beans already commercially available to South Africans.

Anti-nutritional and toxic factors in food legumes: a review.

A comprehensive review on the presence of certain important anti-nutritional and toxic factors in food legumes has been conducted. These substances include proteolytic inhibitors, phytohemagglutinins, lathyrogens, cyanogenetic compounds, compounds causing favism, factors affecting digestibility and saponins. These factors are shown to be widely present in leguminous foods which are important constituents of the diet of a large section of the world's population, and particularly, of people in the developing countries.

Comparison of growth, nitrogen metabolism and organ weights in piglets and rats fed on diets containing Phaseolus vulgaris beans.

The effects of lectins in the diet have been mainly studied in rats. An important question is whether results obtained in rats can be extrapolated to larger animals like the pig. Phaseolus vulgaris beans are rich in toxic lectins. Therefore a study was carried out to compare the effects of diets containing 200 g Phaseolus vulgaris beans (raw or toasted)/kg in rats and piglets. Live-weight gain, nitrogen digestibility and N balance were much lower in piglets than in rats fed on diets containing raw beans. Live-weight gain and N balance were slightly negative in the piglets. When toasted beans were given, live-weight gain and N balance values were reduced in piglets but hardly at all in rats. Giving raw beans caused hypertrophy of the pancreas in the rats but in piglets the weight of the pancreas was reduced. Spleen weight was depressed in the piglets but not in the rats. Weight of liver was not affected in either animal species. When toasted beans were given no effects on the weights of pancreas, spleen or liver were found in piglets or rats. It was concluded that the piglet is much more sensitive to antinutritional factors in the Phaseolus vulgaris bean than the rat.

Anaphylactic shock after skin test with phytohaemoagglutinin.

A young woman (26 years old), with gastrointestinal candidosis of 8 months duration, was hospitalized in our department to study her cell immunity. Five minutes after an id injection of PHA (bactophytohaemoagglutinin M, Difco, 1 mg), mix monilieae, mix tricophyton and PPD Berna, she showed an extensive wheal and flare reaction in the PHA injection area, eyelid oedema and respiratory distress. In spite of adrenaline, corticosteroid and antihistamine drug administration, the patient collapsed and was admitted to the resuscitation department, where she quickly recovered. In the following days, the patient underwent immune and allergy testing: 1) total Ige immuno-enzymatic assay: 72 KU/1; 2) specific IgE and IgE immuno-enzymatic assay for Candida: negative; 3) in vitro lymphocyte transformation test (thymidine 2-C14) with PHA Difco was positive. This finding and the positivity of the id injection with PHA could indicate an allergic type I immunoreaction, the description of which we have not found in the international literature (the presence of IgG STS cannot be totally excluded).

Effect of oral phytohaemagglutinin intake on cell adaptation in the epithelium of the small intestine of the rat.

The effect of phytohaemagglutinin (PHA) on the adaptation of the proximal jejunal epithelium and on the distal ileal epithelium was studied in rats. The group receiving PHA gained less weight than controls, and the enterocyte population of their jejunal villi, as well as the morphokinetic parameters (length, population, crypt cell production per crypt) of their jejunal and ileal crypts were higher than those of the controls. The proximal lesion caused by PHA (reduction of villus cell populations) stimulates hyperplasia of the crypt-villus unit of the ileal epithelium with the development of adaptation from afar. These adaptations occurred in animals that ingested PHA even in the presence of severe malnutrition.

Augmentative effects of phytohemagglutinin-P on proliferation and cytotoxicity of interleukin-2-activated canine peripheral blood lymphocytes.

Canine peripheral blood lymphocytes (PBL) were simulated with recombinant human interleukin-2 (rhIL-2) alone, or with phytohemagglutinin-P (PHA) and subsequent rhIL-2 in order to obtain large numbers of lymphokine-activated killer (LAK) cells. Incubation of PBL with rhIL-2 alone allowed proliferation of large granular lymphocyte (LGL)-like lymphocytes, and the cytotoxic activity of the lymphocytes against canine transmissible venereal sarcoma cells was detected 5-7 days after the culture onset. However, the lymphocytes died within 2 weeks of culture, resulting in limited generation of functional LAK cells. Thus, PBL pretreated with PHA are subjected to rhIL-2-dependent culture. Small- or middle-sized lymphocytes predominantly proliferated in response to rhIL-2, and proliferation of the lymphocytes was sustained for longer than 30 days by repeated stimulations with PHA and subsequent rhIL-2. Cytotoxicity reached significant levels from 2 weeks after the culture onset and thereafter remained almost constant for at least 2 weeks, leading to large-scale production of the LAK cells. Pretreatment of PBL and PHA seems to enhance the LAK cell functions through modification of the precursors of the effector LAK cells.

Effect of variable protein contents in diets containing Phaseolus vulgaris beans on performance, organ weights and blood variables in piglets, rats and chickens.

A comparison was made of the effects of antinutritional factors present in Phaseolus vulgaris on piglets, rats and chickens. Also the hypothesis of whether the negative effect on weight gain due to the inclusion of raw Phaseolus vulgaris in the diet can be attributed to an insufficient supply of amino acids was tested. Test diets containing 200 g raw Phaseolus beans/kg were balanced for digestible protein and amino acids; in one diet extra casein was incorporated. The main response criteria were live-weight gain and the weight of various organs including the intestine. Live-weight gain in piglets was markedly reduced during feeding 200 g raw Phaseolus vulgaris/kg in the diet, but not in rats and chickens. Addition of casein did not improve the weight gain of the piglets, indicating that a toxic factor was responsible for the reduced weight gain and not an insufficient supply of amino acids. The weights of the spleen and thymus were markedly reduced in the piglets when the diets with raw Phaseolus beans were given, but not in the rats and chickens. Additional supply of casein did not change this effect. Indications were found that when the supply of dietary protein is adequate there is no reduction in pancreas weight with raw Phaseolus beans as was observed in previous experiments. The weight of the intestine was increased in all three species due to feeding raw Phaseolus vulgaris.

Morphological and behavioral drawbacks of fetal dopaminergic grafts, prelabeled with Phaseolus vulgaris leucoagglutinin.

The anterograde tracer Phaseolus vulgaris Leucoagglutinin (Pha-L) was tested as a fetal cell marker in short-term labeling of a fetal dopaminergic cell suspension and in long-term surviving grafts in vivo. As a model we used the grafting of fetal dopaminergic cells into the denervated caudate putamen of the rat. Short-term labeling revealed that the viability of the fetal cells was not affected by the Pha-L incubation within the 4 h of the test period. Yet, a subtle difference was noticed in the morphological development of the fetal neurons. Whereas many dopaminergic cells in the control suspension developed from an initially round soma to a more triangular or bipolar one, Pha-L-incubated cells maintained their round appearance. Moreover, cells with developing neurites were commonly noted in the control suspension, but were absent after incubation with Pha-L. Long-term effects of Pha-L were studied in three groups of unilaterally 6-hydroxydopamine-lesioned rats, which all received an injection of a fetal dopaminergic cell suspension in the denervated caudate putamen. The first group (T-Pha-L) received dopaminergic cells, prelabeled with Pha-L. The second group (T-saline) received cells incubated with vehicle (saline). The third group (T) received only dissociated cells. Eight weeks after the implantation the morphological analysis showed a minor Pha-L-immunoreactivity inside the labeled grafts. We detected Pha-L-positive fiber particles as well as weakly Pha-L-positive spots, presumably cell bodies. Pha-L-labeled grafts were significantly decreased in graft volume and contained markedly less dopamine-immunoreactive (DAi) cells than the control grafts of groups T-saline and T. The ratio DAi cell type I (cell with < or = 3 processes)/DAi cell type II (cell with > or = 4 processes) was approximately 8 in the control groups and 3 in group T-Pha-L. This suggests primarily a toxic effect of Pha-L and DAi cell type I neurons. Our behavioral data revealed that the Pha-L-labeled grafts did not cause a recovery from lesion-induced motor asymmetries.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy.

The immune function of retrovirus-mediated gene modified (GM) T cells is critical for a beneficial effect to follow their adoptive transfer into patients. Recent clinical data show that GM T cells expanded with PHA have reduced function in vivo. However, little functional analysis of PHA stimulation is available. Our results show that expansion of T cells with PHA impairs their ability to respond (proliferation, cytotoxicity and IFN gamma and perforin expression) to allogeneic stimulation or viral antigens in vitro. Conversely, CD3/CD28-based protocols can preserve this immune function. Retroviral transduction did not alter the functional profile induced by polyclonal stimulation. We investigated the mechanisms leading to this functional effect, and identified differential effects of PHA and CD3/CD28 on the distribution of CCR7/CD45RA T cell functional subsets, which may explain the functional differences observed. While CD3/CD28 stimulation parallels the lineage differentiation pattern induced by antigens in physiological conditions, PHA induces a skewed distribution of the CCR7/CD45RA functional T cell subsets, with near disappearance of the subpopulations that display the effector phenotype. Overall, this study demonstrates a functional disadvantage for transduction protocols based on PHA, uncovers mechanisms that may explain this functional effect, and provides us with information to design and select transduction protocols with an improved functional outcome.

Kidney bean lectin-induced Escherichia coli overgrowth in the small intestine is blocked by GNA, a mannose-specific lectin.

The reversible and dose-dependent hyperplastic growth of the small intestine and accelerated epithelial cell turnover caused by feeding rats with diets containing kidney bean lectin (PHA) increased the proportion of immature cells on the villi whose membrane and/or cytoplasm contained mainly simple, polymannosylated glycans. These new alpha-linked mannosyl terminals, particularly of the damaged epithelium, facilitated the preferential adherence of opportunistic Escherichia coli with mannose-sensitive Type 1 fimbriae, and other coliforms, to the glycocalyx. Accordingly, the growth of the gut was accompanied by a reversible and PHA dose-dependent overgrowth with E. coli. As expected from their common carbohydrate specificity, the inclusion in the diet of the mannose-specific agglutinin from snowdrop (Galanthus nivalis) bulbs (GNA) significantly reduced the extent of E. coli overgrowth, but abolished neither the growth nor the damage caused by PHA to the small intestine. Thus, GNA and perhaps other mannose-specific lectins, especially when used in a preventive mode, can be used to specifically block the proliferation of Type 1 E. coli in the small intestine.

Non-nutrient bioactive substances of pulses.

Pulses supply many bioactive substances found in minor amounts in food, but which may have significant metabolic and/or physiological effects. These compounds have long been classified as antinutritional factors, but many studies have reconsidered their impact on health. Some could play a role in the prevention of the major diseases of affluent societies. As these compounds can be beneficial or adverse, depending on conditions, an assessment of their various physiological effects is necessary to determine whether they should be preserved or eliminated in each main nutritional situation.