Expression of NOS–2, COX–2 and Th1/Th2 cytokines in migraine

Nitric oxide (NO) probably plays an important role in the pathogenesis of migraine without aura (MWA). As the activation of NO–ergic cascade has been shown to be closely linked to cyclooxygenase pathway and to cause some differences in peripheral blood lymphocyte populations, we investigated if the Th1/Th2 balance in peripheral blood of MWA patients was affected in comparison to controls. Twenty–six MWA patients and 10 healthy controls (C) were enrolled in this study. Blood samples were taken at baseline (T0) and during an induced migraine attack (T1). Total RNA from human peripheral blood lymphocytes (PBLs) was isolated and reverse–transcribed to prepare complementary DNA. COX–2, NOS–2 and β–actin were amplified using PCR. PBLs from patients and controls were stimulated with phorbol 12–myristate 13–acetate plus ionomycin in the presence of brefeldin A. Cells were surface–stained with fluorochrome–conjugated anti–CD3 and anti–CD8 monoclonal antibodies (mAbs) and intracellularly stained with fluorochrome– conjugated anti–IFN–γ or anti–IL–4 mAbs. The level of cytokine expression was analyzed by gating on the CD4+ lymphocyte subset. Th1 and Th2 type cytokines (IFN–γ, IL–2, IL–4) were directly assayed in serum by ELISA. Preliminary results indicate that NOS–2 was upregulated in MWA patients at basal time if compared to controls, whereas after NOD administration NOS–2 was significantly decreased. COX–2 was upregulated in MWA patients at basal time and it had an opposite trend after NOD administration. The homeostatic Th1/Th2 balance defined by the IFN–γ or IL–4 cytokine expression was unchanged in MWA patients in comparison to controls, and NOD administration did not affect that pattern. The cell activation machinery was not altered after mitogenic activation, as shown by CD69 expression level. Cytokine serum levels showed no significant changes in all studied situations. This study confirms the relevance of the NOS/COX system in MWA but, in contrast with previous studies, excludes their effect and activation on peripheral cytokine production. More sophisticated experimental models are needed to investigate the ability of NOS/COX to activate migraine pain.     

Involvement of bcl-2 and bax gene expression in apoptosis and differentiation of the non-tumorigenic murine hematopoietic cell line, 32DC13(G)

32DCl3(G) is an interleukin-3 (IL-3) dependent, non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocyte-colony stimulating factor (G-CSF). This line therefore offers a convenient system to study the expression of genes involved in apoptosis and differentiation. In our experiments we have acquired evidence that during the differentiation pathway, likewise in apoptosis induced by IL-3 deprivation, detectable levels of bax mRNA appear, while bcl-2 expression decreases. These events are under the control of the p53 tumor-suppressor gene. In these cells, an overexpression of exogenous wild-type p53 leads to a decrease in bcl-2 mRNA and to the appearance of box mRNA, which instead is absent in the parental cells growing in IL-3 conditioned medium. Furthermore, results from experiments on p53 transfected cells demonstrate that excess wild-type p53 activity, on its own, fails to elicit apoptosis as long as IL-3 is present and does not induce differentiation if G-CSF is not added to the culture medium. We conclude that in apoptosis and differentiation of 32DCl3(G) the alterate ratio of bcl-2 and box gene expression, modulated by p53, is an early event dependent on IL-3 withdrawal and that the appearance of bax and the decrease of bcl-2 expression are necessary, but not sufficient for the acquisition of a completely mature granulocytic phenotype.     

Defective lymphocyte caspase-3 expression in type 1 diabetes mellitus

OBJECTIVE: Activation-induced cell death (AICD) is a major mechanism in the regulation of peripheral tolerance and its impairment can determine the development of autoimmunity. In the present study, in order to evaluate the role of caspase-3 in type 1 diabetes mellitus (T1DM) AICD, caspase-3 expression was analyzed in peripheral blood lymphocytes from 37 new onset T1DM patients and from 36 normal control subjects (NC) in resting conditions and after anti-Fas-triggered apoptosis. METHODS: Caspase-3 expression was determined by semiquantitative RT-PCR and Western blot. Apoptosis was induced in activated lymphocytes by anti-Fas monoclonal antibody and quantified by flow cytometry and morphological analysis. RESULTS: Caspase-3 mRNA expression was reduced in resting lymphocytes in 18/37 T1DM patients and in 1/36 NC (P < 0.01). Patients studied for both Fas-mediated AICD and caspase-3 mRNA expression revealed that a reduced caspase-3 mRNA expression in resting lymphocytes occurred in all patients showing resistance to Fas-mediated apoptosis (T1DM vs NC, P < 0.02) with the exception of 3 patients who exhibited normal caspase-3 expression levels. Caspase-3 protein analysis confirmed mRNA data and showed an impaired expression of caspase-3 active form in T1DM subjects compared with NC. CONCLUSIONS: Our data show that defective expression and function of caspase-3 in peripheral lymphocytes of T1DM patients may contribute to the development of AICD resistance in type 1 diabetes.     

Estradiol 17-beta and progesterone modulate inducible nitric oxide synthase and high mobility group box 1 expression in human endometrium

The aim of the present study is to investigate the effects of ovarian sex steroid hormones on the expression and the release of several locally active substances by human endometrium. Specific objectives are (1) to ascertain if estradiol 17-beta (E2) and progesterone modulate inducible nitric oxide synthase (iNOS) expression and nitric oxide release; (2) to determine whether human endometrium can express High Mobility Group Box 1 (HMGB1), a multifunctional cytokine, and whether sexual steroid hormones can modulate this expression; and (3) to evaluate whether nitric oxide can influence HMGB1 expression in this tissue. Endometrial tissue was obtained from 40 healthy premenopausal women who underwent hysteroscopy for suspected benign gynecological conditions. Endometrium was incubated with E2, progesterone, or sodium nitroprusside, a nitric oxide donor. Nitrite assay was used to quantify stable nitric oxide metabolites in culture medium, and Western blot analysis was used to detect iNOS and HMGB1. Incubation of endometrium with E2 results in an increase in iNOS expression and nitric oxide metabolite production. The opposite effect is obtained by incubating tissues with progesterone. HMGB1 is expressed by human endometrium, and its expression is increased by E2 and decreased by progesterone. Incubation with sodium nitroprusside results in a reduction in HMGB1 expression. Both E2 and progesterone modulate iNOS expression and nitric oxide production in human endometrium. HMGB1 is expressed in the human endometrium, and its expression is modulated by E2, progesterone, and nitric oxide.