Leukocytosis, muscle damage and increased lymphocyte proliferative response after an adventure sprint race

The effect of an adventure sprint race (ASR) on T-cell proliferation, leukocyte count and muscle damage was evaluated. Seven young male runners completed an ASR in the region of Serra do Espinhaço, Brazil. The race induced a strong leukocytosis (6.22±2.04×103 cells/mm3 beforevs 14.81±3.53×103 cells/mm3after the race), marked by a significant increase of neutrophils and monocytes (P<0.05), but not total lymphocytes, CD3+CD4+ or CD3+CD8+ cells. However, the T-cell proliferative response to mitogenic stimulation was increased (P=0.025) after the race, which contradicted our hypothesis that ASR, as a high-demand competition, would inhibit T-cell proliferation. A positive correlation (P=0.03, r=0.79) was observed between the proliferative response of lymphocytes after the race and the time to complete the race, suggesting that the proliferative response was dependent on exercise intensity. Muscle damage was evident after the race by increased serum levels of aspartate amino transferase (24.99±8.30 vs 50.61±15.76 U/L, P=0.003). The results suggest that humoral factors and substances released by damaged muscle may be responsible for lymphocyte activation, which may be involved in muscle recovery and repair.

Clonal analysis of the BALB/c T cell proliferative response to apo beef cytochrome c.

Murine T cell clones that proliferated specifically in response to the protein antigen apo cytochrome c were derived and maintained in continuous culture. Two distinct clonotypes were observed with respect to the proliferative responses observed when a variety of peptides prepared from several species of cytochrome c were tested. These 2 clonotypes appeared to recognize 2 different regions in the cytochrome c molecule. Only 1 of the 2 clonotypes tested demonstrated helper cell activity for antibody formation in vitro.

Functional analysis of the murine T lymphocyte immune response to a protozoan parasite, Leishmania tropica

The results presented in this review summarize a seirs of experiments designed to characterize the murine T cell imune response to the protozoan parasite Leishmania tropica. Enriched T cell populations and T cell clones specific for L. tropica antigens were derived from lymph nodes of primed mice and maintained in continous culture in vitro. These T lymphocytes were shown (A) to express the Lyt 1+ 3- cell surface phenotype, (B) to proliferate specifically in vitro in response to parasite antigens, together with a source of irradiated syngeneic macrophages, (C) to transfer antigen-specific delayed-type hypersensitivity (DTH) responses to normal syngeneic mice, (D) to induce specific activation of parasitized macrophages in vitro resulting in the destruction of intracellular parasites, (E) to provide specific helper activity for antibody responses in vitro in a hapten-carrier system. Protection studies using these defiened T cell populations should allow the characterization of parasite antigen(s) implicated in the induction of cellular immune responses beneficial for the host.

Cell mediated immunity in Chagas' disease: Trypanosoma cruzi antigens induce suppression of the in vitro proliferative response of mononuclear cells

The partial suppression of the cell-mediated immune response by Trypanosoma cruzi antigens in patients with Chagas' disease is demonstrated in a costimulation assay with T. cruzi antigens and Mycobacterium tuberculosis purified protein derivative (PPD) or Tetanus toxoid (TT). ononuclear cells from 13 patients with chagasic infection without evidence of heart disease, 10 patients with chagasic cardiomyopathy and 7 healthy blood donors were stimulated with antigen A (autoclaved epimastigotes), PPD, TT, PPD + A, PPD + TT and TT + A. The average percentage of suppression induced by costimulation of mononuclear cells with PPD and antigen A was 47.1% in patients with chagasic infection without heart disease (INF), 38.8% in patients with chagasic cardiomyopathy (CDM) and 23.3% in healthy controls. Similar values were observed when living trypomastigotes were used. A costimulatory study with PPD and TT, PPD and A and TT and A was carried out in 8 patients with chagasic infection, in order to evaluate the possibility that this difference could be due to a nonspecific inhibitory effect. The mean suppression induced by TT + PPD was -8.9, with TT + A was 52.7 and with PPD + A was 50.1. The data reported show that T. cruzi antigens induce a specific suppression of the proliferative responseof mononuclear cells, that might be relevant to the persistence of the parasite in the host.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 ± 1.9 mV) compared to NWR (-69 ± 1.4 mV), NWR 1K-1C (-60.8 ± 1.6 mV), WKY (-67.1 ± 3.2 mV) and WKY 1K-1C (-56.9 ± 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 ± 0.86) when compared to arteries of all the other groups (NWR 0.93 ± 0.65, NWR 1K-1C 1.24 ± 0.45, WKY 1.22 ± 0.32, WKY 1K-1C 1.15 ± 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state.

Exposure to parabens at the concentration of maximal proliferative response increases migratory and invasive activity of human breast cancer cells in vitro

Alkyl esters of p–hydroxybenzoic acid (parabens) are widely used as preservatives in personal care products, foods and pharmaceuticals. Their oestrogenic activity, their measurement in human breast tissue and their ability to drive proliferation of oestrogen-responsive human breast cancer cells has opened a debate on their potential to influence breast cancer development. Since proliferation is not the only hallmark of cancer cells, we have investigated the effects of exposure to parabens at concentrations of maximal proliferative response on migratory and invasive properties using three oestrogen-responsive human breast cancer cell lines (MCF-7, T-47-D, ZR-75-1). Cells were maintained short-term (1 week) or long-term (20±2 weeks) in phenol-red-free medium containing 5% charcoal-stripped serum with no addition, 10-8M 17-oestradiol, 1-5x10-4M methylparaben, 10-5M n-propylparaben or 10-5M n-butylparaben. Long-term exposure (20±2 weeks) of MCF-7 cells to methylparaben, n-propylparaben or n-butylparaben increased migration as measured using a scratch assay, time-lapse microscopy and xCELLigence technology: invasive properties were found to increase in matrix degradation assays and migration through matrigel on xCELLigence. Western immunoblotting showed an associated downregulation of E-cadherin and -catenin in the long-term paraben-exposed cells which could be consistent with a mechanism involving epithelial to mesenchymal transition. Increased migratory activity was demonstrated also in long-term paraben-exposed T-47-D and ZR-75-1 cells using a scratch assay and time-lapse microscopy. This is the first report that in vitro, parabens can influence not only proliferation but also migratory and invasive properties of human breast cancer cells.

Differential proliferative response of the ventral prostate and seminal vesicle to testosterone replacement

We have investigated epithelial cell proliferation and the rate of glandular recovery of the ventral prostate (VP) and seminal vesicle (SV) promoted by testosterone replacement (TR) in castration-induced regressed glands. Adult male Wistar rats were castrated and, after 21 days, they were treated with testosterone propionate (4 mg/kg/day). Intact (CT) and castrated rats without TR (CS) were also analysed. VP and SV were processed for histochemistry, morphometric-stereological analysis and immunocytochemistry to determine the PCNA index (PI). After 10 days of TR, the VP weight reached similar to 72% of the CT values, while the SV weight exceeded similar to 17% of the CT values. By the third day of TR, VP and SV presented a mean P1 of 34% and 94% for distal region and 14% and 22% for proximal region, respectively. SV also had more luminal cells PCNA-positive than VP, mainly in the distal region. The PI values fell on days 5, 7 and 10, but were still higher than CT. These findings indicate that epithelial cells from involuted SV are more responsive to TR than those from VP when Stimulated to proliferate and replace the luminal cell population, suggesting a different mechanism regulating cell proliferation in response to androgenic stimuli. (c) 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Alterations of cell-mediated immune response in children with febrile seizures

The aim of the present investigation was to study the distribution of T-cell subsets in peripheral blood defined by monoclonal antibodies and by the lymphocyte proliferative response to phytohemagglutinin (PHA) in 30 children with febrile seizures and in 14 age-matched control subjects. Frequent respiratory, urinary and dermatologic infections were observed in 22 patients. The immunologic parameters showed that 64% of the patients presented an increased number of CD8+ cells and a low helper/suppressor ratio was observed in 60% of the patients. In addition, the proliferative response of lymphocytes to PHA was impaired in the patients It was observed the presence of inhibitory activity on lymphocyte function in the plasma of 33% of children with febrile seizures. These results suggest that patients with febrile seizures have an impairment of cellular immunity that may be connected with this epileptic syndrome and explain the infections observed.

Lymphocyte blastogenic response to ovalbumin in a model for canine allergy

Lymphocyte stimulation tests (LST) were performed in five dogs sensitised with ovalbumin (OVA) and seven healthy dogs. In addition, all five OVA-sensitised and two control dogs were tested after two in vivo provocations with OVA-containing eye drops. The isolated cells were suspended in culture media containing OVA and were cultured for up to 12 days. Proliferation was measured as reduction in 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) intensity by flow cytometry on days 0, 3, 6, 9 and 12. A cell proliferation index (CPI) for each day and the area under the curve (AUC) of the CPI was calculated for each dog. All OVA-sensitised dogs demonstrated increased erythema after conjunctival OVA application. The presence of OVA-specific lymphocytes was demonstrated in 2/5 OVA-sensitised dogs before and 4/5 after in vivo provocation. Using the AUC, the difference between OVA-sensitised and control dogs was significant in all three LST before in vivo provocation (P<0.05) and borderline significant (P=0.053) in 2/3 LST after provocation. The most significant difference in CPI was observed after 9 days of culture (P=0.001). This pilot study indicates that the LST allows detection of rare antigen specific memory T-cells in dogs previously sensitised to, but not concurrently undergoing challenge by a specific antigen.

Effect of Pru p 3 on dendritic cell maturation and T-lymphocyte proliferation in peach allergic patients.

BACKGROUND: Pru p 3 is the major peach allergen and the most frequent cause of food allergy in adults in the Mediterranean area. Although its allergenicity is well characterized, its ability to generate a T-cell response is not completely known. OBJECTIVE: To investigate the influence of Pru p 3 allergen on dendritic cell (DC) maturation and specific T-cell response (T(H)1/T(H)2) in peach allergic patients. METHODS: Peach allergic patients (n = 11) and tolerant controls (n = 14) were included in the study. Monocyte-derived DC maturation after incubation with Pru p 3 was evaluated by the increase of maturational markers (CD80, CD86, and CD83) by flow cytometry. Lymphocyte proliferation was evaluated by coculturing monocyte-derived DCs and 5,6-carboxyfluorescein diacetate N-succinimidyl ester-stained lymphocytes with different concentrations of Pru p 3 (25, 10, and 1 ?g/mL) by flow cytometry and cytokine production. RESULTS: Pru p 3 induced a significant increase in the CD80, CD86, and CD83 expression on stimulated DCs from patients compared with controls. The lymphocyte proliferative response after Pru p 3 stimulation was also significantly higher along with an increase in interleukin 8 in patients compared with tolerant controls. CONCLUSION: Pru p 3 allergen induces changes in DC maturational status mainly in peach allergic patients. An increase in lymphocyte proliferative response accompanied with a different cytokine pattern was also observed compared with healthy controls.

Interferon β transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens

We are developing a gene therapy method of HIV infection based on the constitutive low production of interferon (IFN) β. Peripheral blood lymphocytes (PBL) from HIV-infected patients at different clinical stages of infection were efficiently transduced with the HMB-HbHuIFNβ retroviral vector. The constitutive low production of IFN-β in cultured PBL from HIV-infected patients resulted in a decreased viral production and an enhanced survival of CD4+ cells, and this protective effect was observed only in the PBL derived from donors having a CD4+ cell count above 200 per mm3. In IFN-β-transduced PBL from healthy and from HIV-infected donors, the production of the Th1-type cytokines IFN-γ and interleukin (IL)-12 was enhanced. In IFN-β-transduced PBL from HIV-infected donors, the production of IL-4, IL-6, IL-10, and tumor necrosis factor α was maintained at normal levels, contrary to the increased levels produced by the untransduced PBL. The proliferative response to recall antigens was partially restored in IFN-β-transduced PBL from donors with an impaired antigen response. Thus, in addition to inhibiting HIV replication, IFN-β transduction of PBL from HIV-infected donors improves several parameters of immune function.

Differential proliferative response of NKT cell subpopulations to in vitro stimulation in presence of different cytokines

Human Valpha24(+)Vbeta11(+) NKT (NKT) cells have immune regulatory activities associated with rejection of tumors, infections and control of autoimmune diseases. They can be stimulated to proliferate using alpha-galactosylceramide (KRN7000) and have the potential for therapeutic manipulation. Subpopulations of NKT cells (CD4(+)CD8(-), CD4(-)D8(+) and CD4(-)CD8(-)) have functionally distinctive Th1/Th2 cytokine profiles and their relative numbers following stimulation may influence the Th1/Th2 balance, which may result in or prevent disease. We aimed to determine the effect of different cytokines in culture during stimulation of NKT cells on the relative proportions of NKT cell subpopulations. Our results show that all NKT cell subpopulations expanded following stimulation with KRN7000 and IL-2, IL-7, IL-1 2 or IL-15. Expansion capacity differed between subpopulations, resulting in different relative proportions of CD4(+) and CD4(-) NKT cell subpopulations, and this was influenced by the cytokine used for stimulation. A Th1-biased environment was observed after stimulation of NKT cells. NKT cells expanded under all conditions evaluated demonstrated significant cytotoxicity against U937 tumor cells. In view of the potential for NKT cell subsets to alter the balance of Th1 and Th2 environment, these data provide insights into the effects of NKT cell manipulation for possible therapeutic applications in different disease settings.

TGF-beta and CD23 are involved in nitric oxide production by pulmonary macrophages activated by beta-glucan from Paracoccidioides brasiliensis

Pulmonary macrophages (PM), which are CD11b/CD18(+) and CD23(+), may be involved in the onset of inflammatory events caused by Paracoccidioides brasiliensis in the lungs. In the present study, we measured the nitric oxide (NO) and interleukin in PM production after intratracheal (i.t.) inoculation of an enriched beta-glucan cell wall fraction from P. brasiliensis (Fraction F1). BALB/c and C57/BL6 (B6) mice were i.t. treated with Fraction F1, and their PM were restimulated in vitro with LPS and interferon-gamma up to 14 days after treatment. Macrophages BALB/c mice produced less NO than PM from B6 mice. The lower NO production was caused by higher production of TGF-beta by pulmonary macrophages of BALB/c and was abrogated by anti-TGF-beta MoAb in vitro and in vivo. Other interleukins such as IL-10, IL-4 and a combination of IL-1, TNF-alpha and IL-6 were not involved in NO production induced by Fraction F1. Expression of CD11b increases and expression of CD23 decreases on PM of BALB/c mice after in vivo treatment whereas PM of B6 mice do not show a variation of their phenotype. Moreover, the ability of pulmonary macrophages to induce lymphocyte proliferation was reduced in mixed cultures of CD11b(+) or CD23(+) macrophages but was restored when lymphocytes were cultivated in the presence of NO inhibitor (L-NMMA). Thus, the results presented herein indicate that in BALB/c but not in B6 mice TGF- is strongly induced by Fraction 1 in PM in vivo and suppresses NO production. Low NO production by PM is associated with a change in CD11b/CD23 expression and with a high lymphocyte proliferative response. Thus, CD11b(+)/CD23(+) PM modulate NO and TGF-beta production in the pulmonary microenvironment.

Impact of Antifibrotic Treatment on the Course of Schistosoma mansoni Infection in Murine Model

Administration of an antifibrotic agent as an adjunct to antihelmintic treatment with the objective of morbidity reduction was investigated in the murine schistosomiasis mansoni model. Antifibrotic, ß-aminopropionitrile treatment has a profound effect on the cellular matrix composition of the liver granuloma of Schistosoma mansoni infected mice when given alone, resulting in increase macrophage infiltration. These macrophages, in response to stimulation with soluble egg antigen or lipopolysaccharide produced elevated levels of nitric oxide but low levels of tumor necrosis factor alpha compared to untreated infected mice. This also correlated with reduced liver granuloma size. In spite of low numbers of eggs in the liver, mice receiving a combine treatment had a high level of resistance to a challenge infection compared with mice receiving only praziquantel. Those mice also exhibited a reduced lymphocyte proliferative response, similar to that of infected untreated mice. Antifibrotic treatment has an impact on the dynamic of the cellular nature of granulomas and impacts on the host immunity to infection

Infliximab partially impairs the anti-Mycobacterium tuberculosis immune responses of severe psoriasis patients with positive tuberculin skin-test

Background Infliximab and etarnecept are now widely used for treating severe psoriasis. However, these drugs, especially infliximab, increased the risk of tuberculosis reactivation. Surprisingly, epidemiological data suggest that the tuberculosis rate in patients taking infliximab in Sao Paulo State, Brazil, is similar to that of some developed, non-endemic countries. Objective The aim of this study was to better understand the effect of infliximab on Mycobacterium tuberculosis (Mtb) immune responses of psoriasis patients in an endemic setting (Brazil). Methods We evaluated the tuberculosis-specific immune responses of severe psoriasis patients and healthy individuals, both tuberculin skin test (TST) positive, in the presence/absence of infliximab. Patients had untreated severe psoriasis, no co-morbidities affecting the immune responses and a TST >10 mm. Healthy TST+ (>10 mm) individuals were evaluated in parallel. PBMC cultures from both groups were stimulated with different Mycobacterium tuberculosis (Mtb) antigens (ESAT-6, 85B and Mtb lysate) and phytohemagglutinin, with or without infliximab (5 mu g/mL). Parameters evaluated were TNF-alpha, IFN-gamma and IL-10 secretion by ELISA, overnight IFN-gamma ELISpot and lymphocyte proliferative response (LPR). Results Infliximab almost abolished TNF-alpha detection in PBMC supernatants of both groups. It also significantly reduced the LPR to phytohemagglutinin and the Mtb antigens as well as the IFN-gamma levels secreted into day 5 supernatants in both groups. There was no concomitant exaggerated IL-10 secretion that could account for the decreases in these responses. ELISpot showed that, contrasting with the central-memory responses above, infliximab did not affect effector-memory INF-gamma-releasing T-cell numbers. Conclusions Infliximab affected some, but not all aspects of the in vitro antituberculosis immune responses tested. The preserved effector-memory responses, putatively related to exposure to environmental mycobacteria, may help to explain the lower than expected susceptibility to tuberculosis reactivation in our setting. Received: 29 December 2010; Accepted: 9 March 2011