Generation and characterization of two human alpha/beta T cell clones. Recognizing autologous breast tumor cells through an HLA- and TCR/CD3-independent pathway.

Cell-mediated immune response to breast tumor has only been marginally investigated. To gain insight into this issue we have developed two clones of distinct phenotype, CD3+ alpha/beta, CD4+, CD8-, CD16-, and CD3+ alpha/beta, CD4-, CD8+, CD16-, respectively, from peripheral blood lymphocytes (PBL) of a breast cancer patient. These effectors, selected on the basis of their cytolytic activity against autologous tumor cells and lack of lysis on NK-sensitive cell lines, preferentially recognize autologous tumor cells. The two clones' cytotoxic activity, while inhibited by anti-LFA-1 mAb, could not be abolished by mAbs to CD3, to class I and class II MHC molecules, and by mAbs to molecules involved in T cell function (i.e., CD4, CD8, CD2). The molecular structure of the alpha and beta T cell receptor chains of the two effector cells, confirmed their clonality and showed that, despite an overlapping killing pattern, they possess distinct TCR alpha and beta chains. These findings demonstrate that breast tumor-specific CTL clones can be generated through current technology and that a alpha/beta effector cell population operating through a HLA-unrestricted and TCR/CD3-independent pathway may be involved in the identification and killing of this tumor.

[Crystalline silica can induce oxidative stress by inhibiting glyoxalase system in bronchial epithelial cells]. / La silice cristallina (Min-U-Sil 5) induce stress ossidativo inibendo l'espressione del sistema delle gliossalasi in cellule bronchiali umane (BEAS-2B).

UNLABELLED: Chronic inflammation and reactive oxygen species (ROS) production induced by crystalline silica are involved in the development of silicosis and lung cancer pathogenesis. ROS can generate lipid peroxydation of cell membranes that can produce methylglyoxal (MG), a strong cell proliferation inhibitor and apoptosis inducer. MG is naturally removed by glyoxalase I (GI) and glyoxalase II (GII) through a glutathione (GSH) dependent mechanism. Therefore mRNA expression of glyoxalases is correlated to MG concentration and oxidative stress. OBJECTIVES: evaluate oxidative stress induced by crystalline silica by glyoxalases mRNA expression and methylglyoxal concentration MATERIAL AND METHODS: In bronchial epithelial cell culture (BEAS-2B), exposed to 50 microg/cm2 crystalline silica (Min-U-Sil 5), for 2, 6, 12, and 24 hours, GI and GII mRNA levels and MG intracellular concentration were measured respectively by Real-Time PCR and HPLC. RESULTS: Crystalline silica exposure induced a significant reduction in mRNA expression of glyoxalases and an increase of MG intracellular concentration. CONCLUSIONS: The results suggest a possible use of MG and mRNA expression of GI and GII as crystalline silica induced oxidative stress indicators.

Activated platelets and leucocytes cooperatively stimulate smooth muscle cell proliferation and proto-oncogene expression via release of soluble growth factors.

BACKGROUND: Previous studies indicate that platelets and leucocytes might contribute to the development of neointimal hyperplasia following arterial injury. The present study was aimed at further investigating the role of platelets and leucocytes, alone or in combination, in promoting vascular smooth muscle cell (SMC) proliferation in vitro, focusing on the relative contribution of different soluble growth factors released by these cells, and on the ability to induce proto-oncogene expression, such as c-fos. METHODS: SMCs from rabbit aortas, made quiescent by serum deprivation, were stimulated with either activated platelets, leucocytes, or both, separated from SMCs by a membrane insert. SMC proliferation was evaluated by measuring the incorporation of 3H-thymidine. The relative contribution of different platelet-derived mediators to SMC growth was evaluated by adding either ketanserin, a 5-HT2 receptor antagonist, R68070, a TxA2 receptor antagonist, BN52021, a platelet activating factor (PAF) receptor antagonist, and trapidil, a platelet derived growth factor (PDGF) receptor antagonist. The role of different leucocyte sub-populations (neutrophils and monocytes + lymphocytes) was also determined in additional experiments. RESULTS: SMC proliferation was significantly increased by activated platelets to 360 +/- 9% of control values (P < 0.05). This effect was reduced by ketanserin, R68070, BN 52021 or trapidil. Whole leucocytes, neutrophils or lymphocytes + monocytes also increased SMC proliferation with respect to control experiments. Simultaneous stimulation of SMCs by platelets and whole leucocytes was associated with a significant greater increase in SMC proliferation as compared to SMC stimulated with platelets or leucocytes alone. c-fos expression, almost undetectable in unstimulated SMCs, was markedly increased by activated platelets or leucocytes. CONCLUSIONS: Activated platelets promote SMC proliferation in vitro via release of soluble mediators, including serotonin, thromboxane A2 PAF and PDGF; activated leucocytes also induce a significant SMC proliferation and exert an additive effect when activated together with platelets; SMCs stimulated with activated platelets and leucocytes show an early expression of the proto-oncogene c-fos.

Immediate early genes and brain DNA remodeling in the Naples high- and low-excitability rat lines following exposure to a spatial novelty.

The aim of these studies was to map the neural consequences of exposure to a spatial novelty on the expression of immediate gene (IEG) and on unscheduled brain DNA synthesis (UBDS) in two genetic models of altered activity and hippocampal functions, i.e., the Naples High- (NHE) and Low-excitability (NLE) rats. Adult male rats of NLE and NHE lines, and of a random-bred stock (NRB) were tested in a Làt-maze, and corner crossings, rearings, and fecal boli were counted during two 10-min tests 24 h apart. For IEG expression, rats were exposed to a Làt-maze with nonexposed or repeatedly exposed rats used as controls, and were sacrificed at different time intervals thereafter. For UBDS, rats were sacrificed immediately after the first or the second exposure o a Làt-maze. IEG expression was measured by immunocytochemistry for the FOS and JUN proteins. NRB rats exposed for the first time to the maze showed extensive FOS and JUN positive cells in the reticular formation, the granular and pyramidal neurons of hippocampus, the amygdaloid nuclei, all layers of somatosensory cortex, and the granule cells of the cerebellar cortex. The positivity, stronger in rats exposed for the first time, was present between 2 and 6 h and was prevented by the NMDA receptor antagonist CPP (5 mg/kg). The positivity was very low in NHE rats, and it was stronger in NLE compared to NRB rats. UBDS was measured in ex vivo homogenates of brain areas by the incorporation into DNA of 3H-[methyl]-thymidine given intraventricularly 15 min before test trial 1 or 2 (pulse of 0.5 h).(ABSTRACT TRUNCATED AT 250 WORDS)

The dorsal noradrenergic bundle modulates DNA remodeling in the rat brain upon exposure to a spatial novelty.

A series of experiments were designed to study the role of the dorsal noradrenergic bundle (DNB) in the modulation of genomic remodeling in the mammalian brain. A series of experiments were designed to study the role of the dorsal noradrenergic system in relation to nonassociative tasks. Adult male Sprague-Dawley rats were either bilaterally lesioned in the DNB by intrabundle microinjection of 6-hydroxydopamine or were sham lesioned. All rats were given 50 microCi [3H-methyl]-thymidine and were sacrificed 0.5 h later. After the injection of the tracer, rats were either left undisturbed in the home cage or were exposed to a Làt-maze for 15 min after 15 min had passed from the time of injection. During the exposure to the maze, corner crossings and rearings were monitored. The rate of DNA synthesis was determined in several brain regions by measuring the amount of tracer incorporated into the DNA over a 0.5-h duration pulse. Under baseline conditions DNB-lesioned rats showed an increase in DNA synthesis in the hippocampus, hypothalamus, and rest of the brain. On the other hand, following exposure to the Làt-maze, sham-lesioned rats only showed an increase in DNA synthesis in the hippocampus, as compared to baseline conditions. Conversely, DNB-lesioned rats did not show an increase in hippocampal DNA synthesis as in the sham-lesioned rats. In contrast, DNA synthesis was increased in the neocortex and rest of the brain. In conclusion, the data support a role for noradrenergic systems in modulating brain DNA synthesis, probably of the unscheduled type, during information processing and storage.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic receptor systems and unscheduled DNA synthesis in the rat brain.

Two experiments were carried out in the albino rat to investigate the role of brain adrenergic systems in DNA remodeling. Adult male Sprague-Dawley rats were given an intraventricular microinjection of an adrenergic drug or vehicle followed 2 h later by the intraventricular injection of 50 microCi of [3H-methyl]thymidine. The rats were sacrificed 0.5 h after the injection of the radioactive tracer. The rate of DNA synthesis was determined by measuring the amount of radioactive precursor incorporated into the DNA extracted from homogenates of several brain areas. In Experiment 1, at time 0 rats received the alpha-adrenergic antagonist phentolamine (5 micrograms), the beta antagonist propranolol (10 micrograms), the alpha agonist phenylephrine (1 microgram), the beta agonist isoproterenol (12.5 micrograms), or the vehicle. The latter decreased UBDS in neocortex, and increased it in the septum, neostriatum, hypothalamus, cerebellum, and rest of the brain. The alpha and beta agonists and antagonists induced several significant effects, depending on the brain region. In Experiment 2, rats were bilaterally lesioned in the dorsal noradrenergic bundle (DNB) by injection of 6-hydroxydopamine or were sham lesioned. One week later, at time 0 they were given the alpha agonist phenylephrine (1 microgram), the beta agonist isoproterenol (12.5 micrograms), or the vehicle. The DNB-lesioned rats showed a higher UBDS in the hippocampus, neocortex, and hypothalamus, which was reversed by the alpha or the beta agonist. The results suggest an influence of the DNB, probably as a tonic inhibitor of UBDS in the hippocampus and the hypothalamus which, in turn, are likely to be mediated by beta- and alpha-adrenergic receptors. In addition, a phasic inhibitory effect seems to be mediated by beta and alpha receptors in the neocortex, and by beta receptors in the cerebellum. A modulatory role of central adrenergic systems on unscheduled brain DNA synthesis may be inferred from these findings.

Slow epidemic extinction in populations with heterogeneous infection rates.

We explore how heterogeneity in the intensity of interactions between people affects epidemic spreading. For that, we study the susceptible-infected-susceptible model on a complex network, where a link connecting individuals i and j is endowed with an infection rate ß(ij)=λw(ij) proportional to the intensity of their contact w(ij), with a distribution P(w(ij)) taken from face-to-face experiments analyzed in Cattuto et al. [PLoS ONE 5, e11596 (2010)]. We find an extremely slow decay of the fraction of infected individuals, for a wide range of the control parameter λ. Using a distribution of width a we identify two large regions in the a-λ space with anomalous behaviors, which are reminiscent of rare region effects (Griffiths phases) found in models with quenched disorder. We show that the slow approach to extinction is caused by isolated small groups of highly interacting individuals, which keep epidemics alive for very long times. A mean-field approximation and a percolation approach capture with very good accuracy the absorbing-active transition line for weak (small a) and strong (large a) disorder, respectively.

Design and evaluation of a wireless electronic health records system for field care in mass casualty settings.

BACKGROUND: There is growing interest in the use of technology to enhance the tracking and quality of clinical information available for patients in disaster settings. This paper describes the design and evaluation of the Wireless Internet Information System for Medical Response in Disasters (WIISARD). MATERIALS AND METHODS: WIISARD combined advanced networking technology with electronic triage tags that reported victims' position and recorded medical information, with wireless pulse-oximeters that monitored patient vital signs, and a wireless electronic medical record (EMR) for disaster care. The EMR system included WiFi handheld devices with barcode scanners (used by front-line responders) and computer tablets with role-tailored software (used by managers of the triage, treatment, transport and medical communications teams). An additional software system provided situational awareness for the incident commander. The WIISARD system was evaluated in a large-scale simulation exercise designed for training first responders. A randomized trial was overlaid on this exercise with 100 simulated victims, 50 in a control pathway (paper-based), and 50 in completely electronic WIISARD pathway. All patients in the electronic pathway were cared for within the WIISARD system without paper-based workarounds. RESULTS: WIISARD reduced the rate of the missing and/or duplicated patient identifiers (0% vs 47%, p<0.001). The total time of the field was nearly identical (38:20 vs 38:23, IQR 26:53-1:05:32 vs 18:55-57:22). CONCLUSION: Overall, the results of WIISARD show that wireless EMR systems for care of the victims of disasters would be complex to develop but potentially feasible to build and deploy, and likely to improve the quality of information available for the delivery of care during disasters.

Nucleo-cytoplasmic interactions in the petite negative yeast Schizosaccharomyces pombe. Inhibition of nuclear and mitochondrial DNA syntheses in the absence of cytoplasmic protein synthesis.

In the petite positive yeast, Saccharomyces cerevisiae, cycloheximide selectively inhibits protein synthesis on cytoplasmic ribosomes, and, as a consequence, nuclear DNA synthesis. Mitochondrial DNA, however, is synthesized for 4-6 h after cessation of protein synthesis. In this paper we show that in contrast to Saccharomyces cerevisiae, synthesis of mitochondrial and nuclear DNA is tightly coordinated in the petite negative yeast Schizosaccharomyces pombe, since inhibition of cytoplasmic protein synthesis leads immediately to cessation of both nuclear and mitochondrial DNA synthesis.

Ultrasonographic follow-up of patients with choroidal melanoma following conservative treatment.

The authors assessed the regression of choroidal tumors, following irradiation treatment, by means of B scan sonography (Sonomed B 3000). Thirty-two patients were studied, 12 of whom underwent brachytherapy with 106Ru plaques and 20 of whom were treated with accelerated protons. After a follow-up period of 12 months, the following was observed: reduction of the thickness of the tumor (significantly greater in the tumors which underwent brachytherapy) and morphological and structural changes which consisted in a thinning of the tumor and an increased reflectivity.

Graves' disease: measurement of the extraocular muscle thickness with the echobiometer.

The authors measured extraocular muscle thickness in normal subjects and in patients affected by Graves' disease, using a Sonomed A-2000 echobiometer (probe with 10-MHz frequency); Hertel's exophthalmometry was also performed. Statistically significant differences in muscle thickness between normals and patients were found. This technique seems to be sufficiently useful and reliable in extraocular thickness evaluation, showing data similar to those of the recent literature.

Long-term culture and T cell receptor analysis of T cell clones isolated from a patient with adenosine deaminase deficiency and type I diabetes.

We performed limiting dilution culture of T cells from a patient affected by primary immunodeficiency as a result of complete lack of adenosine deaminase (ADA) activity and also affected by insulin-dependent diabetes mellitus (type I diabetes). Despite the occurrence of immunodeficiency, we were able to raise and grow T cell clones derived from this patient in long-term culture. These T cells displayed ADA enzymatic activity and produced interleukin-2 after engagement of their T cell receptor (TCR)/CD3 complex. We analyzed the TCR repertoire of such clones by nucleotide sequencing of TCR beta chains. The results show that the T cell clones express different V beta but similar J regions. However, the CDR3 regions which are implicated in antigen recognition were found to be heterogeneous.

Emerging antithrombotic treatments for acute coronary syndromes.

In the last few years the hypothesis of coronary thrombosis, frequently triggered by plaque ulceration or fissuration, has gained wide acceptance as one of the key events in the pathophysiology of acute coronary syndromes. Plaque ulceration may activate both platelets and the coagulation cascade via exposure of a variety of substances, such as von Willebrand factor and tissue factor. It has been demonstrated that aspirin reduces mortality and improves the prognosis of patients with such syndromes. More recently, newer drugs have been identified for the treatment of acute coronary syndromes; in particular, platelet glycoprotein IIb/IIIa inhibitors have been found to be more effective than aspirin in a variety of clinical conditions, such as unstable angina, acute myocardial infarction, and coronary angioplasty. Other drugs with different mechanisms of action will be soon available for large scale clinical trials.

Characterization of a dodecapeptide containing a dominant epitope of Par j 1 and Par o 1, the major allergens of P. judaica and P. officinalis pollen.

The pollen of Parietaria, a weed of the Urticaceae family, is a major cause of respiratory allergy in Europe, where the most common species are P. judaica and P. officinalis. Previously, we reported that a beta-galactosidase fusion protein (6a-BG) expressing a 26-bp cDNA fragment (6a cDNA) contained a dominant IgE-binding epitope (6a epitope) of the major allergens Par o 1 and Par j 1. The present study aimed to define the amino-acid sequence containing the 6a epitope. We analyzed the reactivity of anti-Par o 1 antibodies affinity purified from allergic patient sera with: 1) a panel of synthetic peptides deduced from the 6a nucleotide sequence using different reading frames 2) glutathione S-transferase (GST) fusion proteins containing selected peptides. The peptide NSARARADSCRI (p102) specifically bound anti-Par o 1 antibodies affinity purified from allergic patient sera or from rabbit anti-Par o 1 antiserum (ELISA). The related peptide NSARAGTSSCRI (p101) reacted to human but not to rabbit, anti-Par o 1 antibodies. GST fusion proteins containing p101 (GST 3.5) or p102 (GST 3.2) extensively inhibited the binding between Par o 1 and IgE or IgG antibodies from an allergic patient serum pool according to a dose-response curve. Percent inhibition of IgE antibodies binding obtained by absorbing a solution (50 microl) of affinity-purified antibodies with 5 microg of GST 3.2 or with 1.2 mg of GST 3.5 was 69% and 66%, respectively. In conclusion, the results of the present study indicate that the amino-acid sequences NSARARADSCRI (p102) and NSARAGTSSCRI (p101) contain the dominant epitope of Par o 1 and Par j 1 for human IgE and IgG antibodies indicated as 6a epitope. Moreover, the study shows that the epitope is conserved in recombinant molecules containing these peptides, irrespective of the fused polypeptide (beta-galactosidase or GST). The knowledge of the amino-acid sequence of this dominant epitope is important in therapeutic approaches to the development of allergen-derived haptens.

Cross-reactivity between the major Parietaria allergen and rotavirus VP4 protein.

BACKGROUND: The present study investigates immunological cross-reactivity between Par o 1, the major pollen allergen of Parietaria, and the VP4 protein of rotavirus, a microorganism that is world-wide the main etiological agent of gastroenteritis in children. METHODS: IgG and IgE cross-reactivity was assessed by direct binding and competitive inhibition assays (ELISA and DARIA), using recombinant VP4 from rhesus infectious rotavirus (RR), synthetic peptides and Par o 1-specific antibodies affinity purified from pooled and individual human sera. RESULTS: Antibodies specifically binding Par o 1, affinity purified from the sera of 35 individuals with skin test positivity to Parietaria and from 14 pools, were extensively cross-reactive with RRVP4. Cross-reactive binding was specifically inhibited by synthetic peptides derived from the C-terminal sequences of the VP4 proteins from human and rhesus infectious rotavirus. CONCLUSIONS: This study reports the first evidence of cross-reactivity between an allergen and a viral antigen.

Antithrombotic effects of recombinant human, active site-blocked factor VIIa in a rabbit model of recurrent arterial thrombosis.

The extrinsic coagulation pathway is activated when circulating factor VII (FVII) gains access to tissue factor (TF) exposed as a consequence of vascular injury. Increasing evidence indicates that this TF-dependent activation of the coagulation plays an important role in the pathophysiology of intravascular thrombus formation. In the present study, we tested the effects of recombinant human, active site-blocked activated FVII (FVIIai) in a rabbit model of carotid artery thrombosis. Cyclic flow variations (CFVs), due to recurrent thrombus formation, were obtained in stenotic rabbit carotid arteries with endothelial injury. Carotid blood flow velocity was measured by a Doppler flow probe. After 30 minutes of CFVs, the animals received FVIIai (100 microg x kg(-1) x min(-1) intracarotid infusion for 10 minutes, n=9). If CFVs were abolished, animals were followed for 30 additional minutes, after which recombinant human activated FVII (FVIIa) was infused into the carotid artery (100 microg x kg(-1) x min(-1) for 10 minutes) to determine whether FVIIai could be displaced from TF by FVIIa, thus restoring CFVs. To establish the duration of action of FVIIai, an additional group of animals received FVIIai at the same dose as above, and after CFVs were inhibited, they were followed until CFVs were restored or for up to 6 hours. To determine whether CFVs could be restored by epinephrine after their abolition with FVIIai, increasing doses of epinephrine were administered to a third group of 6 animals. FVIIai abolished CFVs in 8 of 9 rabbits (P<.01). This effect was reversible, as FVIIa administration restored CFVs in all animals. Prothrombin times and activated partial thromboplastin times did not change significantly throughout the study. One single 10-minute infusion exerted complete antithrombotic effects for at least 6 hours, despite the fact that at this time point, plasma FVIIai levels were well below threshold concentrations. Epinephrine restored CFVs in 3 of 6 animals in which CFVs were inhibited by FVIIai. FVIIai exerts potent antithrombotic effects in this model; these effects were prolonged even after FVIIai was almost completely cleared from the circulation, probably as a result of the tight binding of FVIIai to TF. Thus, FVIIai might represent an antithrombotic substance of potential interest.

[Effect of platelets and leukocytes on in vitro proliferation of muscle cells: role of platelet-derived chemical mediators]. / Effetti delle piastrine e dei leucociti sulla proliferazione delle cellule muscolari in vitro: ruolo dei mediatori chimici di derivazione piastrinica.

Some evidence indicate that platelets (PLTs) and leukocytes might contribute to the development of neointimal hyperplasia following arterial injury, via release of several growth factors. To study the relative contribution of these cells and of growth factors released in consequence of activation, smooth muscle cells (SMCs), isolated from the aorta of New Zealand White rabbits, were grown in Dulbecco's medium containing 10% fetal calf serum (FCS). At 70% confluence, SMCs were made quiescent by removing FCS from the medium. Twenty-four hours later, the cells were stimulated with activated platelets, neutrophils, lymphocytes+monocytes, whole leukocytes and platelets + whole leukocytes. Then, 1 microCi of O3H-thymidine were added to SMC cultures to evaluate the degree of proliferation. Relative contribution of different PLT-derived mediators to SMC growth was evaluated by adding either ketanserin, a 5-HT2 receptor antagonist, ridogrel, a thromboxane A2 (TxA2) receptor antagonist, BN52021, a platelet activating factor (PAF) receptor antagonist, and trapidil, a platelet-derived growth factor (PDGF) receptor antagonist, or all antagonists together. SMC proliferation was significantly increased by platelet activation. This effect was reduced by adding either ketanserin, ridogrel, BN 52021 or trapidil. Neutrophils, lymphocytes + monocytes and whole leukocytes also increased SMC proliferation. Simultaneous stimulation of SMCs by platelets and whole leukocytes was associated with a significant increase in SMC proliferation as compared to platelets or leukocytes alone. Thus, TxA2, 5-HT, PAF, and PDGF all contribute to SMC proliferation in vitro. Adding all antagonist together resulted in an additive antiproliferative effect. Leukocytes are also important in SMC proliferation. Interaction between platelets and leukocytes may play a pivotal role in the modulation of this phenomenon.

Recombinant human, active site-blocked factor VIIa reduces infarct size and no-reflow phenomenon in rabbits.

Oxygen free radicals induce de novo synthesis of tissue factor (TF), the initiator of the extrinsic pathway of coagulation, within the coronary vasculature during postischemic reperfusion. In the present study we wanted to assess whether TF expression might cause myocardial injury during postischemic reperfusion. Anesthetized rabbits underwent 30 min of coronary occlusion followed by 5.5 h of reperfusion. At reperfusion the animals received 1) saline (n = 8), 2) human recombinant, active site-blocked activated factor VII (FVIIai, 1 mg/kg, n = 8), or 3) human recombinant activated FVII (FVIIa, 1 mg/kg, n = 8). FVIIai binds to TF as native FVII, but with the active site blocked it inhibits TF procoagulant activity. The area at risk of infarction (AR), the infarct size (IS), and the no-reflow area (NR) were determined at the end of the experiment. FVIIai resulted in a significant reduction in IS and NR with respect to control animals (28.1 +/- 11.3 and 11.1 +/- 6.1% of AR vs. 59.8 +/- 12.8 and 24.4 +/- 2.7% of AR, respectively, P < 0.01), whereas FVIIa resulted in a significant increase in IS and NR to 80.1 +/- 13. 1 and 61.9 +/- 13.8% of AR, respectively (P < 0.01). In conclusion, TF-mediated activation of the extrinsic coagulation pathway makes an important contribution to myocardial injury during postischemic reperfusion.