Sensitivity to cisplatin-induced mutations and elevated chromosomal aberrations in lymphocytes from sickle cell disease patients

Sickle cell disease (SCD) is an inherited disorder caused by a single nucleotide substitution in the P-globin gene. The clinical heterogeneity observed in SCD patients has been attributed to environmental and genetic factors. The patients are subjected to increased oxidative stress, particularly during vaso-occlusive crises and acute chest pain. Another possible cause of oxidative stress in SCD is the high concentration of iron in the patients` plasma. The increase in oxidative stress could be a relevant risk factor for mutagenesis and carcinogenesis. Studies on the frequency of basal chromosomal aberrations in cultured lymphocytes from SCD patients have not been reported so far. In order to contribute to the understanding of the role of the different biomarkers and their relationship with the extremely variable clinical manifestation of SCD, we investigated the frequency of chromosome damage in peripheral lymphocytes from sickle cells patients and healthy controls. We found an increased frequency of chromosome damage and percentage of aberrant metaphases in these patients when compared with control subjects, even at basal values (p < 0.05). In the cytogenetic sensitivity assay, the results showed that these patients presented a marked decrease in the mitotic index values compared with healthy controls. Cisplatin-induced chromosomal damage in lymphocytes from these patients was significantly higher than the frequency measured in healthy controls. The results obtained in the present study showed that more investigations are needed in order to elucidate the susceptibility to genomic instability of SCD patients.

Evaluation of the genotoxicity of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes

In the present study, experiments were carried out to evaluate the mutagenic potential and genotoxic effects of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes, using the micronucleus and comet assays. Significant damage to DNA was observed for crotoxin and crotapotin (CA). Basic phospholipase A(2) (CB) and crotamine did not present any mutagenic potential when evaluated by the micronucleus test. C. d. terrificus crude venom was able to induce the formation of micronuclei, similarly to the mutagenic drug used as a positive control. In the comet assay, all the toxins tested (crotamine, crotoxin, CB and CA) and C. d. terrificus venom presented genotoxic activity. Studies on the cytogenetic toxicology of animal venoms and their isolated proteins are still very scarce in the literature, which emphasizes the importance of the present work for the identification and characterization of potential therapeutic agents, as well as for the better understanding of the mechanisms of action of toxins on the human body. (C) 2011 Elsevier B.V. All rights reserved.

The azo dyes Disperse Red 1 and Disperse Orange 1 increase the micronuclei frequencies in human lymphocytes and in HepG2 cells

The use of azo dyes by different industries can cause direct and/or indirect effects oil human and environmental health due to the discharge of industrial effluents that contain these toxic compounds. Several studies have demonstrated the genotoxic effects of various azo dyes, but information on the DNA damage caused by Disperse Red 1 and Disperse Orange 1 is unavailable, although these dyes are used in dyeing processes in many countries. The aim of the present study was to evaluate the mutagenic activity of Disperse Red 1 and Disperse Orange 1 using the micronucleus (MN) assay in human lymphocytes and in HepG2 cells. In the lymphocyte assay. it was found that the number of MN induced by the lowest concentration of each dye (0.2 mu g/mL) was similar to that of the negative control. At the other concentrations, a dose response MN formation was observed up to 1.0 mu g/mL. At higher dose levels, the number of MN decreased. For the HepG2 cells the results were similar. With both dyes a dose dependent increase in the frequency of MN was detected. However for the HepG2, the threshold for this increase was 2.0 mu g/mL, while at higher doses a reduction in the MN number was observed. The proliferation index was also calculated in order to evaluate acute toxicity during the test. No differences were detected between the different concentrations tested and the negative control. (C) 2009 Elsevier B.V. All rights reserved.

Anticlastogenic and antigenotoxic effects of selenomethionine on doxorubicin-induced damage in vitro in human lymphocytes

The use of antioxidants during chemotherapy has been shown to reduce or prevent the undesirable effects experienced by healthy cells. Micronutrient selenium is well known for its antioxidant properties; however, selenium exhibits a bimodal nature in that both its beneficial and toxic properties lie within a limited and narrow dose range. The present study investigated the possible protective effects of selenomethionine (SM) on the cytotoxicity, genotoxicity and clastogenicity of the chemotherapic doxorubicin (DXR), a key chemotherapic used in cancer treatment. Human peripheral lymphocytes were treated in vitro with varying concentrations of SM (0.25 mu M, 0.5 mu M, 1.0 mu M and 2.0 mu M), tested in combination with DXR (0.15 mu g/mL). SM alone was not cytotoxic and when combined with DXR treatment, reduced the DNA damage index significantly, the frequency of chromosomal aberrations, the number of aberrant metaphases and the frequency of apoptotic cells. The mechanism of chemoprotection of SM may be related to its antioxidant properties as well as its ability to interfere with DNA repair pathways. Therefore this study showed that SM is effective in reducing the genetic damage induced by the antitumoral agent DXR. (C) 2007 Elsevier Ltd. All rights reserved.

Evaluation of the cytogenetic effects of I-131 preceded by recombinant human thyrotropin (rhTSH) in peripheral lymphocytes of Wistar rats

The present study was carried out to investigate the cytogenetic effects of therapeutic exposure to radioiodine preceded by rhTSH in an animal model. Three groups of Wistar rats (n = 6) were used: one group was treated only with I-131 (11.1 MBq/animal); the other two groups received rhTSH (1.2 mu g/rat of either Thyrogen or rhTSH-IPEN, respectively) 24 h before administration of radioiodine. The percentage of lymphocytes with chromosome aberrations and the average number of aberrations and of dicentrics per cell were determined on blood samples collected 24 h, 7 and 30 days after administration of I-131. The data show that the treatment with radioiodine alone or associated with rhTSH resulted in a greater quantity of chromosome alterations in relation to basal values after 24 h, with a gradual decline after 7 and 30 days of treatment. An increase in chromosome alterations was also seen after rhTSH treatment alone. Neither of the treatments, i.e., with I-131 alone or associated with hormone, resulted in an aneugenic effect or influenced the kinetics of cellular proliferation in rat blood lymphocytes. There was no significant difference between the cytogenetic effects of Thyrogen and rhTSH-IPEN treatment. These data suggest that the treatment with radioiodine, associated or not with rhTSH, affects to a limited extent a relatively small number of cells although the occurrence of late stochastic effects could not be discarded.

Mesenchymal stromal cells up-regulate CD39 and increase adenosine production to suppress activated T-lymphocytes

Mesenchymal stromal cells (MSCs) suppress T cell responses through mechanisms not completely understood. Adenosine is a strong immunosuppressant that acts mainly through its receptor A(2a) (ADORA2A). Extracellular adenosine levels are a net result of its production (mediated by CD39 and CD73), and of its conversion into inosine by Adenosine Deaminase (ADA). Here we investigated the involvement of ADO in the immunomodulation promoted by MSCs. Human T lymphocytes were activated and cultured with or without MSCs. Compared to lymphocytes cultured without MSCs, co-cultured lymphocytes were suppressed and expressed higher levels of ADORA2A and lower levels of ADA. In co-cultures, the percentage of MSCs expressing CD39, and of T lymphocytes expressing CD73, increased significantly and adenosine levels were higher. Incubation of MSCs with media conditioned by activated T lymphocytes induced the production of adenosine to levels similar to those observed in co-cultures, indicating that adenosine production was mainly derived from MSCs. Finally, blocking ADORA2A signaling raised lymphocyte proliferation significantly. Our results suggest that some of the immunomodulatory properties of MSCs may, in part, be mediated through the modulation of components related to adenosine signaling. These findings may open new avenues for the development of new treatments for GVHD and other inflammatory diseases. (C) 2011 Elsevier B.V. All rights reserved.

Transcriptional Response of Peripheral Lymphocytes to Early Fibrosarcoma: A Model System for Cancer Detection Based on Hybridization Signatures

Since circulating leukocytes, mainly B and T cells, continuously maintain vigilant and comprehensive immune surveillance, these cells could be used as reporters for signs of infection or other pathologies, including cancer. Activated lymphocyte clones trigger a sensitive transcriptional response, which could be identified by gene expression profiling. To assess this hypothesis, we conducted microarray analysis of the gene expression profile of lymphocytes isolated from immunocompetent BALB/c mice subcutaneously injected with different numbers of tumorigenic B61 fibrosarcoma cells. Flow cytometry demonstrated that the number of circulating T (CD3(+)CD4(+) or CD3(+)CD8(+)) or B (CD19(+)) cells did not change. However, the lymphocytes isolated from tumor cell-injected animals expressed a unique transcriptional profile that was identifiable before the development of a palpable tumor mass. This finding demonstrates that the transcriptional response appears before alterations in the main lymphocyte subsets and that the gene expression profile of peripheral lymphocytes can serve as a sensitive and accurate method for the early detection of cancer. Exp Biol Med 234:802-812, 2009

Genotoxic effect of Physalis angulata L. (Solanaceae) extract on human lymphocytes treated in vitro

Physalis angulata L (Solanaceae) is a medicinal plant from North of Brazil, whose different extracts and infusions are commonly used in the popular medicine for the treatment of malaria, asthma, hepatitis, dermatitis and rheumatism. However, the genotoxic effects of P. angulata on human cells is not well known. The main purpose of the present study was to evaluate the in vitro genotoxic effects of aqueous extract of P angulata using the comet assay and the micronucleus assay in human lymphocytes provided from 6 healthy donors. Treatments with P angulata extracts were performed in vitro in order to access the extent of DNA damage. The comet assay has shown that treatments with P angulata at 0.5, 1.0, 2.0, 3.0 and 6.0 mu g/mL in Culture medium were genotoxic. Lymphocytes treated with P angulata at the concentrations of 3.0 and 6.0 mu g/mL in culture medium showed a statistically significant increase in the frequency of micronucleus (p<0.05), however, the cytokinesis blocked proliferation index (CBPI) was not decreased after P angulata treatment. In conclusion, the present work demonstrated the genotoxic effects of P angulata extract on human lymphocytes in vitro.

Non-coding transcript in T cells (NTT): Antisense transcript activates PKR and NF-kappa B in human lymphocytes

T cell activation is a complex process involving many steps and the role played by the non-protein-coding RNAs (ncRNAs) in this phenomenon is still unclear. The non-coding T cells transcript (NTT) is differentially expressed during human T cells activation, but its function is unknown. Here, we detected a 426 m NTT transcript by RT-PCR using RNA of human lymphocytes activated with a synthetic peptide of HIV-1. After cloning, the sense and antisense 426 nt NTT transcripts were obtained by in vitro transcription and were sequenced. We found that both transcripts are highly structured and are able to activate PKR. A striking observation was that the antisense 426 nt NTT transcript is significantly more effective in activating PKR than the corresponding sense transcript. The transcription factor NF-kappa B is activated by PKR through phosphorylation and subsequent degradation of its inhibitor I-kappa B beta. We also found that the antisense 426 nt NTT transcript induces more efficiently the degradation Of I-kappa B beta than the sense transcript. Thus, this study suggests that the role played by NTT in the activation of lymphocytes can be mediated by PKR through NF-kappa B activation. However, the physiological significance of the activity of the antisense 426 nt NTT transcript remains unknown. (c) 2007 Elsevier Inc. All rights reserved.

Chronic colitis associated with HIV infection can be related to intraepithelial infiltration of the colon by CD8+T lymphocytes

Gastrointestinal complications in AIDS patients with diarrhoea are common clinical manifestations, frequently diagnosed by colonoscopy as non-specific colitis. We retrospectively study colon biopsies diagnosed as chronic colitis associated with HIV (CCH). Biopsies were sorted as patients with AIDS (serum CD4 < 200 cell/mm(3)) but without any clear infectious process (n = 12) and patients without HIV infection (n = 24). There are low numbers of CD4+ T lymphocytes in lamina propria of AIDS patients, but CD8+ T populations in this area appear to be similar in all studied groups, regardless of HIV infection or laboratory evidence of a specific agent. We found the clear evidence of CD8+ T cells infiltration in colonic mucosa in HIV patients with microscopic colitis. An imbalance of lymphocyte subpopulations in the colon, both in the lamina propria and epithelium, could result in an intraepithelial CD8 infiltration, involved in the pathogenesis of CCH in AIDS patients.

Glucose metabolism by lymphocytes, macrophages, and tumor cells from Walker 256 tumor-bearing rats supplemented with fish oil for one generation

Here we investigated the effect of lifelong supplementation of the diet with coconut fat (CO, rich in saturated fatty acids) or fish oil (170, rich in n-3 polyunsaturated fatty acids) on tumor growth and lactate production from glucose in Walker 256 tumor cells, peritoneal macrophages, spleen, and gut-associated lymphocytes. Female Wistar rats were supplemented with CO or FO prior to mating and then throughout pregnancy and gestation and then the male offspring were supplemented from weaning until 90 days of age. Then they were inoculated subcutaneously with Walker 256 tumor cells. Tumor weight at 14 days in control rats (those fed standard chow) and CO supplemented was approximately 30 g. Supplementation of the diet with FO significantly reduced tumor growth by 76%. Lactate production (nmol h(-1) mg(-1) protein) from glucose by Walker 256 cells in the group fed regular chow (W) was 381.8 +/- 14.9. Supplementation with coconut fat (WCO) caused a significant reduction in lactate production by 1.6-fold and with fish oil (WFO) by 3.8-fold. Spleen lymphocytes obtained from W and WCO groups had markedly increased lactate production (553 +/- 70 and 635 +/- 150) when compared to non-tumor-bearing rats (similar to 260 +/- 30). FO supplementation reduced significantly the lactate production (297 +/- 50). Gut-associated lymphocytes obtained from W and WCO groups increased lactate production markedly (280 +/- 31 and 276 +/- 25) when compared to non-tumor-bearing rats (similar to 90 +/- 18). FO supplementation reduced significantly the lactate production (168 +/- 14). Lactate production by peritoneal macrophages was increased by tumor burden but there was no difference between the groups fed the various diets. Lifelong consumption of FO protects against tumor growth and modifies glucose metabolism in Walker tumor cells and lymphocytes but not in macrophages. Copyright (C) 2008 John Wiley & Sons, Ltd.

Lymphocytes transfer [(14)C]-labeled fatty acids to skeletal muscle in culture; modulation by exercise

Previous studies have shown that lipids are transferred from lymphocytes (Ly) to different cell types including macrophages. enterocytes, and pancreatic beta cells in co-culture This study investigated whether [(14)C]-labeled fatty acids (FA) can be transferred from Ly to skeletal muscle (SM), and the effects of exercise on such phenomenon Ly obtained from exercised (EX) and control (C) male Wistar rats were preloaded with the [(14)C]-labeled free FA palmitic (PA), oleic (OA), linoleic (LA), or arachidonic (AA) Radioactively loaded Ly were then co-cultured with SM from the same Ly donor animals Substantial amounts of FA were transferred to SM being the profile PA = OA > AA > LA to the C group. and PA > OA > LA > AA to the EX group These FA were incorporated predominantly as phospholipids (PA = 66 75%: OA = 63 09%, LA = 43 86%, AA - 47 40%) in the C group and (PA = 63 99% OA = 52 72%, LA = 55 99%, AA = 63 40%) in the EX group Also in this group, the remaining radioactivity from AA, LA, and OA acids was mainly incorpoiated in structural and energetic lipids These results support the hypothesis that Ly are able to export lipids to SM in co-culture Furthermore. exercise modulates the lipid transference profile, and its incorporation on SM The overall significance of this phenomenon in vivo remains to be elucidated. Copyright (C) 2010 John Wiley & Sons, Ltd

Effect of doxorubicin on cytokine production by lymphocytes and the Th1/Th2 balance

Doxorubicin (DOXO) is a potent chemotherapeutic used mainly against solid tumours; however, it has several side effects that can limit its clinical use. On the other hand, the effect of DOXO upon lymphocyte function is controversial. Some studies demonstrate that DOXO administration in vitro suppresses T-cell activation, while the cellular function has been shown to increase in vitro. The objective of this study was to investigate the effect of DOXO on lymphocyte cytokine production in rats. The animals were divided into: SAL (control, n = 10) and DOX (DOXO treated, n = 10). The DOX group received only one DOXO dose at 15 kg Kg(-1) by intraperitoneal injection. Forty-eight hours after DOXO administration, the animals were killed by decapitation. IL-2 production was significantly enhanced (p < 0.05) in lymphocytes from rats treated with DOXO (169.17 +/- 21.73 pg mL 10(5) cell) as compared to cells from SAL (45.92 +/- 10.53 pg mL 10(5) cell). The administration of DOXO decreased (<0.05) IL-4 production in the DOXO group (29.85 +/- 13.09 pg mL 10(5) cell) relative to the SAL group (75.08 +/- 15.31 pg mL 10(5) cell). The IL-2/IL-4 ratio was higher (<0.05) in the DOX group (5.99 +/- 0.44), as compared to SAL group (0.73 +/- 0.12). In conclusion, our results suggest that a dose of DOXO promotes an alteration in the Th1/Th2 balance, promoting a shift towards a Th1-dominant cytokine response. (C) 2010 Elsevier Masson SAS. All rights reserved.

Caracterização imunofenotípica e avaliação quantitativa do infiltrado linfocítário junto às áreas de displasia epitelial da próstata de cães adultos sexualmente intactos

O estudo da próstata canina tem se tornado comum em razão da grande incidência de doenças prostáticas nessa espécie e das similaridades com as alterações apresentadas pela glândula prostática humana. Frente à alta frequência de displasias epiteliais acompanhadas de infiltrado linfocitário intersticial e atrofia acinar na espécie canina, o presente estudo teve como objetivos a caracterização imunofenotípica e a avaliação quantitativa desse infiltrado, utilizando marcadores para identificação de linfócitos T (anti-CD3) e B (anti-CD79a). Foram catalogadas 42 lesões displásicas classificadas em discreta (48%), moderada (38%) e acentuada (14%). O infiltrado linfocitário intersticial periacinar junto às áreas de epitélio prostático displásico constituiu-se predominantemente por linfócitos T (66%) e houve interação entre o grau histológico da displasia e o marcador imunoistoquímico, com oscilação na quantidade de células T e B intersticiais em função do grau da displasia epitelial.

Evolução dos doentes com citologia oncótica alterada e colposcopia anal normal

A citologia anal vem sendo usada para rastreamento do carcinoma anal e suas lesões precursoras nas populações de risco. Quando o raspado do canal anal mostra alterações citológicas está indicada o exame com colposcópio e ácido acético para identificar e realizar biópsia para confirmar o achado. Poucos estudos mostram o seguimento dos doentes tratados de condilomas acuminados perianais. Temos usado os métodos em associação e encontrado lesões subclínicas em metade dos doentes, cujo exame proctológico não revelava doença HPV induzida. Essas lesões são tratadas com tópicos. Entretanto, algumas citologias estavam alteradas e a colposcopia anal não revelou doença HPV induzida. O objetivo deste estudo foi observar o comportamento dessas lesões no seguimento semestral, durante 12 meses, e avaliar se a periodicidade da reavaliação foi suficiente para evitar o aparecimento das lesões de alto grau ou superior. Encontramos 58 (21%) entre 273 doentes nessas condições. As reavaliações de 22 deles após um ano mostraram que as colposcopias permaneceram normais em 17 (74%), sendo que em cinco (22%) a citologia voltou aos padrões normais e 12 (52%) persistiram com alterações. Os outros seis (26%) desenvolveram lesões clínicas ou subclínicas provocadas pelo HPV. As contagens de linfócitos T CD4 dos doentes HIV-positivos foram inferiores nos doentes cujas lesões progrediram. Os resultados permitiram concluir que as alterações podem progredir ou regredir neste grupo distinto de doentes, sendo relacionada à imunidade, e que o intervalo de seis meses é suficiente para cada reavaliação.