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The problem of estimating the thermal state of an induction cooking system is analyzed. The first step is to model the thermal system composed by the cooktop, the pot, and the pot content. Then, by relying on the formulated model, the aim is to design a suitable state observer, so that an estimation of the temperature of the cooking vessel content can be made online. Two kinds of observers are proposed. An Extended Kalman Filter (EKF) and a Sliding Mode Observer (SMO). A comparison of the performances which can be obtained with these two solutions is then made by relying on experimental results obtained on a real induction cooktop heating a pot containing a certain quantity of water.

Human hematopoietic stem cells (HSCs) exposed to cytokines in vitro rapidly divide and lose their characteristic functional properties presumably due to the alteration of a genetic program that determines the properties of an HSC. We have attempted to reverse the silencing of this HSC genetic program by the sequential treatment of human cord blood CD34+ cells with the chromatin-modifying agents, 5-aza-2′-deoxycytidine (5azaD) and trichostatin A (TSA). We determined that all CD34+CD90+ cells treated with 5azaD/TSA and cytokines after 9 days of incubation divide, but to a lesser degree than cells exposed to only cytokines. When CD34+CD90+ cells that have undergone extensive number of cell divisions (5-10) in the presence of cytokines alone were transplanted into immunodeficient mice, donor cell chimerism was not detectable. By contrast, 5azaD/TSA-treated cells that have undergone similar numbers of cell divisions retained their marrow repopulating potential. The expression of several genes and their products previously implicated in HSC self-renewal were up-regulated in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. These data indicate that HSC treated with chromatin-modifying agents are capable of undergoing repeated cell divisions in vitro while retaining their marrow-repopulating potential.

Recurrent cytogenetic abnormalities in leukemic blasts make these an attractive source for dendritic cells (DC) to induce a leukemia‐specific immune response. In this study, three leukemic cell lines were investigated: Kasumi‐1 and SKNO‐1 (two acute myeloid leukemia (AML) cell lines carrying the (8;21)‐chromosomal translocation, resulting in the expression of the leukemia‐specific fusion protein AML1‐eight‐twenty‐one) and REH, an acute lymphoblastic leukemia cell line with the (12;21)‐chromosomal translocation and expression of translocation ETS‐like leukemia‐AML1. These fusion proteins are implicated in the pathogenesis of the leukemic state by recruiting corepressors and histone deacetylases (HDAC), which interfere with normal cell differentiation. In vitro generation of DC was achieved using a cytokine cocktail containing tumor necrosis factor α, granulocyte macrophage‐colony stimulating factor, c‐kit ligand, and soluble CD40 ligand; yet, addition of the HDAC inhibitor (Hdi) trichostatin A enhanced DC differentiation with retention of the fusion transcripts. These leukemic DC showed high‐level CD83 and human leukocyte antigen (HLA)‐DR expression and had a high allostimulatory potential. Only DC generated from these cell lines after Hdi induced blast‐specific cytotoxic T cell responses in HLA‐A‐matched T cells with a cytotoxicity of 42% in parental Kasumi‐1 and 83% in parental REH cells, respectively. This model system suggests that the Hdi supports the in vitro differentiation of DC from leukemic blasts with AML1‐containing fusion proteins.

MEF, a recently identified member of the E74 family of ETS-related transcription factors, is a strong transcriptional activator of cytokine gene expression. Using a green fluorescent protein gene reporter plasmid regulated by an MEF-responsive promoter, we determined that the transcriptional activity of MEF is largely restricted to the G1phase of the cell cycle. MEF-dependent transcription was suppressed by the expression of cyclin A but not by cyclin D or cyclin E. This effect was due to the kinase activity generated by cyclin A expression, as co-expression of the cyclin-dependent kinase inhibitors p21 or p27, or a dominant negative form of CDK2 (DNK2), abrogated the reduction of MEF transcriptional activity by cyclin A. Cyclin A-CDK2 phosphorylated MEF protein in vitromore efficiently than cyclin D-CDK4 or cyclin E-CDK2, and phosphorylation of MEF by cyclin A-CDK2 reduced its ability to bind DNA. We determined one site of phosphorylation by cyclin A-CDK2 at the C terminus of MEF, using mass-spectrometry; mutation of three serine or threonine residues in this region significantly reduced phosphorylation of MEF by cyclin A and reduced cyclin A-mediated suppression of its transactivating activity. These amino acid substitutions also reduced the restriction of MEF activity to G1. Phosphorylation of MEF by the cyclin A-CDK2 complex controls its transcriptional activity during the cell cycle, establishing a novel link between the ETS family of proteins and the cell cycle machinery.

ABSTRACTHuman cytomegalovirus (HCMV) UL25 has recently been found to encode a new structural protein that is present in both virion and defective viral particles (C. J. Baldick and T. Shenk, J. Virol. 70:6097–6105, 1996). In the present work a polyclonal antibody was raised against a prokaryotic pUL25 fusion protein in order to investigate the biosynthesis and localization of the UL25 product (pUL25) during HCMV replication in human fibroblasts. Furthermore, pUL25 was transiently expressed in its native form and fused to the FLAG epitope, in COS7 and U373MG cells, in order to compare the properties of the isolated protein and that produced during infection. Immunoblotting analysis revealed a group of polypeptides, ranging from 80 to 100 kDa, in both transfected and infected cells; in vivo labeling experiments with infected cells demonstrated they are posttranslationally modified by phosphorylation. The transcriptional analysis of the UL25 open reading frame combined with the study of pUL25 biosynthesis showed true late kinetics for this protein in infected human fibroblasts. By indirect immunofluorescence both recombinant and viral pUL25 were detected exclusively in the cytoplasm of transfected or infected cells. Interestingly, pUL25 was shown to localize in typical condensed structures in the perinuclear region as already observed for other HCMV tegument proteins. Colocalization of ppUL99 in the same vacuoles suggests that these structure are endosomal cisternae, which are proposed to be a preferential site of viral particle envelopment. Our data suggest that pUL25 is most likely a novel tegument protein and possibly plays a key role in the process of envelopment.

Ten cluster headache patients and 10 healthy controls were subjected to electrocardiographic and pupillometric procedures in a search for cardiac and pupillary sympathergic asymmetry. Sympathergic stimulation was provoked by hyperventilation and by instilling tyramine into both eyes. In the control group, hyperventilation changed neither the T-wave form and polarity nor the QTc. Tyramine provoked an equal mydriasis on the two sides. In cluster headache sufferers, hyperventilation produced changes in the T-wave form and polarity as well as an increase of the QTc due to a disproportionate shortening of the R-R and Q-T intervals. An unequal mydriasis was noted after tyramine instillation due to less marked response on the symptomatic side. The observed electrocardiographic abnormalities are considered an expression of an asynchronous repolarization attributed to a sympathergic asymmetry. It is postulated that both the cardiac and pupillary sympathetic imbalance associated with cluster headache are central in origin.

ABSTRACTß2.7 is the major early transcript produced during human cytomegalovirus infection. This abundantly expressed RNA is polysome associated, but no protein product has ever been detected. In this study, a stable peptide of 24 kDa was produced in vitro from the major open reading frame (ORF), TRL4. Following transient transfection, the intracellular localization was nucleolar and the expression was posttranscriptionally inhibited by the 5' sequence of the transcript, which harbors two short upstream ORFs.

We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition of virus yield in infected-cell supernatants at two weeks postinfection, while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells.

The pressor responsiveness to phenylephrine, an almost pure agonist of peripheral alpha-1-adrenoceptors, was studied in 32 migraine patients in headache-free intervals. Eighteen healthy volunteers served as a control group. Fourteen patients and 14 controls also underwent the tilt test. The following observations were made: (1) the pressor response to phenylephrine was significantly greater and longer lasting in patients than in controls; (2) the reflex decrease of heart rate did not differ in the two groups; (3) a normal orthostatic increase of blood pressure and heart rate occurred in migraineurs with hyperresponsiveness to phenylephrine. These findings suggest a supersensitivity of vascular adrenoceptors which is compatible with a chronic adrenergic deficiency in migraineurs. Since patients did not show an orthostatic hypotension in attack-free periods, the compensatory character of receptoral supersensitivity and the possible mechanisms of cardiovascular dysautonomia, which may occur during migraineous attack, were discussed.

Forty-five patients with cluster headache in the asymptomatic phase were studied by electronic pupillography, testing autonomic function of both pupils pharmacologically. Topical sympathetically-acting mydriatics, tyramine and cocaine and the cholinoceptor blocker, homatropine, induced defective mydriatic responses on the symptomatic side, indicating latent impairment of sympathetic function. The abnormality was found in interattack intervals of the cluster period or during intercluster phases. The tyramine test can be proposed for objective diagnosis of cluster headache. We postulate that cluster attacks are triggered and lateralized by a permanent latent unilateral sympathetic dysfunction. Lithium reduced the mydriatic response to tyramine of the pupil contralateral to the pain, thus restoring the equilibrium between both pupils; this therapy may correct the asymmetric sympathetic function by attenuating the activity in the asymptomatic side.

We have previously shown that single linear epitopes of the major human cytomegalovirus (HCMV) antigens, expressed as fusion proteins or synthesized as oligopeptides, can be valuable diagnostic material in the serology of HCMV infection (M. P. Landini, M. X. Guan, G. Jahn, W. Lindenmaier, M. Mach, A. Ripalti, A. Necker, T. Lazzarotto, and B. Plachter, J. Clin. Microbiol. 28:1375-1379, 1990; M. P. Landini, T. Lazzarotto, A. Ripalti, M. X. Guan, and M. La Placa, J. Clin. Microbiol. 27:2324-2327, 1989; A. Ripalti, M. P. Landini, E. S. Mocarski, and M. La Placa, J. Gen. Virol. 70:1247-1251, 1989). In this work we addressed the question of whether the expression of more than one linear epitope on a single fusion protein could increase the reactivity of genetically engineered antigenic material with human antibody. To answer this question we fused sequences expressing two different epitopes contained in the basic phosphoprotein of 150 kDa encoded by UL32 (M. S. Chee, A. T. Bankier, S. Beck, R. Bohni, C. M. Brown, T. Cerny, T. Hornsel, C. A. Hutchinson, T. Kouzarides, J. A. Martignetti, and B. G. Barrell, Curr. Top. Microbiol. Immunol. 154:125-169, 1990; G. Jahn, T. Kouzarides, M. Mach, B.-C. Scholl, B. Plachter, B. Traupe, E. Preddie, S. C. Satchwell, B. Fleckenstein, and B. G. Barrell, J. Virol. 61:1358-1367, 1987), ppUL32, which was repeatedly shown to be the strongest immunogen present in the viral particle. We also made fusions with sequences expressing a single epitope repeated once, twice, or three times. The different fusion proteins were tested with HCMV-positive human sera. We found that fusion proteins expressing different epitopes together were recognized by a larger number of serum specimens and with more intense reactions in Western blot (immunoblot) experiments. We also found evidence that expression on the same polypeptide of the two distinct epitopes produced a stronger antigen than the mere addition of two fusion proteins which each carried one copy of one of these epitopes. Furthermore, we found that while the same epitope expressed two or three times on the same fusion protein was not better recognized by immunoglobulin G than the single epitope, immunoglobulin M reactivities to the double and triple epitopes were enhanced.

Pupil size was measured using a pupillograph, and an asymmetric responsiveness to tyramine, instilled bilaterally, was observed in asymptomatic cluster headache patients. Relatives of cluster headache patients showed an anisocoric mydriasis to tyramine, too. This asymmetry was caused by a less marked mydriatic response of one side which, in the cluster headache sufferers, corresponds to the symptomatic side. After three months of treatment with lithium carbonate (900 mg/die), a bilateral decrease of pupil size was noted, possibly due to a reduced sympathergic tone. After six months of continued treatment an unknown mechanism, likely adaptive in nature, attenuated the effect of lithium on pupil size. Lithium also induced a symmetric response to tyramine by increasing tyramine mydriasis on the symptomatic pupil while reducing it on the asymptomatic pupil. It is postulated that lithium improves cluster headache by correcting abnormal bilateral asymmetries in central neuronal systems which regulate autonomic function and pain sensitivity of the structures involved in the cluster attack.Au moyen d'études pupillographiques dans la céphalée en chapelet, en phase asymptomatique, on a observé une réponse asymétrique à la tyramine instillée dans les deux yeux. Les membres des familles des patients souffrant de céphalée en chapelet la tyramine révèle une mydriase anisocorique. Cette asymétrie est imputable à une réponse mydriatique moins intense d'un côté qui, chez les patients souffrant de céphalée en chapelet correspond au côté de la douler. Après un traitement de trois mois au carbonate de lithium (900 mg/jour) on a observée una diminution du diamètre de la pupille, vraisemeblablement due à une réduction du ton sympathique. Après six mois de traitment, un mécanisme indéfini, probablement lié à des phénomènes d'adaptation, a fait baisser l'effect myotique du lithium. De plus, le lithium a rendu symétrique la réponse à la tyramine tout en augmentand la mydriase provoquée par la tyramine dans la pupille du côté douloureux et en la faisant abaisser du côté non douloureux. On avance l'hypothèse que dans la céphalée en chapelet le lithium corrige les asymétries anomales de côté à travers son action sur les centres nerveux réglant la fonction autonome et algésiogéne dans les structures empliquées dans l'attaque.Mediante rilevazioni pupillografiche, nella cefalea a grappolo, in fase asintomatica, è stata osservata una risposta asimmetrica alla tiramina instillata in entrambi gli occhi. Anche i familiari dei pazienti con cefalea a grappolo hanno mostrato una midriasi anisocorica da tiramina. Tale asimmetria è dovuta a una meno intensa risposta midriatica da un lato che, nei pazienti con cefalea a grappolo, corrisponde al lato del dolore. Dopo 3 mesi di trattamento con carbonato di litio (900 mg al giorno) si è osservata una diminuzione del diametro pupillare da entrambi i lati, verosimilmente dovuta a un ridotto tono simpatico. Dopo 6 mesi di trattamento un meccanismo non definito, probabilmente legato a fenomeni di adattamento, ha attenuato l'effetto miotico del litio. Il litio inoltre ha reso simmetrica la risposta alla tiramina, aumentando la midriasi da tiramina nella pupilla dal lato colpito e diminuendola dal lato esente dal dolore. Si prospetta che nella cefalea a grappolo il litio corregga le anomale asimmetrie di lato agendo sui centri nervosi che regolano la funzione autonoma e dolorifica nelle strutture coinvolte nell'attacco.

In migraine patients the effects of calcium antagonists (flunarizine, verapamil and nifedipine) on both venous and pupillary neuromuscular functions, as well as on blood pressure have been evaluated. A single oral dose of flunarizine (10 mg) and verapamil infusion (50 μg/ml/min) increased venous compliance. Verapamil also counteracted dose-dependent dopamine induced venoconstriction. Nifedipine (10 mg orally) reduced mean arterial pressure in upright position in migraineurs but not in controls. In addition, chronic treatment with flunarizine (10 mg for 2 weeks) induced a transient miotic effect and a reduction of tyramine induced mydriasis. These findings demonstrated that calcium antagonists affect vascular and extra-vascular structures. It is postulated that, in migraine, calcium entry blockers may prevent exaggerated responses to catecholaminergic stimulation.

The aim of this study was to evaluate the usefulness of serologic analysis for the diagnosis of active cytomegalovirus infection in patients with AIDS. Active cytomegalovirus infection was diagnosed by virus isolation from urine and saliva and detection of antigenemia. Serologic analysis was done by several conventional and innovative procedures. The results indicate no correlation between any of the most popular serologic procedures and virus detection by culture in urine or saliva or antigenemia.

Airborne ultrafine particles (UFPs) potentially have adverse health effects for the exposed population both in life and work environments. Main objective of this study is to propose a strategy to measure multiple parameters that may influence human exposure to UFPs and apply it in a case study at the university site of "La Sapienza" in Rome (Italy). The measurements allow the characterization of UFPs in terms of particle number concentration (PNC), particle size distribution (PSD), lung deposited surface area (LDSA), geometric mean diameter (GMD) and polycyclic aromatic hydrocarbons concentration (PAHs), using data collected in indoor and outdoor conditions by real-time instruments with high time resolution. The study design includes: i) inter-comparison of devices based on different principles of operation to harmonize the outputs in relation to a reference instrument; ii) case study site description and sampling points identification to allow indoor vs outdoor relations at different height; iii) extensive measurement campaigns to provide a dataset useful to data comparison; iv) statistical data series analysis. The preliminary results reported here are related to the values of PNC and calculated average diameter (Davg) for airborne UFPs, obtained in three different sampling points of the research workplaces at the university site: auditorium (about 300 places - ground floor), computer science laboratory (about 50 workstations - first floor) and external roof (top of the building). Major relations between PNC and Davg have been highlighted in daily trends.

Human hematopoietic stem cells (HSCs) exposed to cytokine combinations in vitro rapidly divide and lose their characteristic functional properties presumably due to the alteration of a genetic program which determines the properties of HSC. In order to expand the number of HSC present in a single unit of cord blood (CB) ex vivo, self-renewal type of HSC division must occur. We hypothesize that in vitro culture conditions result in the silencing of genes crucial for HSC maintenance and that silencing of these genes can be circumvented by addition of chromatin modifying agents. We have attempted to reverse the silencing of the genes crucial for HSC self-renewal which apparently occurs during the ex vivo culture by treatment of CD34+ cells with the chromatin modifying agents, 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA). In our current studies, we have investigated the mechanism of expansion of SRC following treatment with chromatin modifying agents in the culture. We demonstrate that all CD34+CD90+ cells treated with 5azaD/TSA and cytokines after 9 days of incubation divide, but to a lesser degree than cells exposed to cytokines alone. CD34+CD90+ cells exposed to the chromatin modifying agents are capable of producing greater numbers of primitive multipotential progenitors and also form cobblestone areas. When CD34+CD90+ cells that had undergone extensive number of cell divisions (5–10) in vitro in the presence of cytokines alone were re-isolated by FACS and transplanted into immunodeficient mice, donor cell chimerism was not detectable (0 of 5 mice). By contrast, 5azaD/TSA treated cells that had undergone similar numbers of cell divisions retain their marrow repopulating potential (3 of 6 mice). To test whether chromatin modifying agents treated cells following culture possess long-term in vivo repopulation potential, we have performed secondary NOD/SCID assay. Five of six secondary NOD/SCID mice receiving bone marrow from primary mice engrafted with cells treated with 5azaD/TSA resulted in human cell engraftment, indicating that these cells are capable of secondary reconstitution. To understand the molecular mechanism responsible for the expansion of HSC observed following 5azaD/TSA treatment, we examined transcription levels of several genes and their products (i.e., HOXB4, Bmi-1 and P21) implicated in self-renewal of HSC using real-time quantitative PCR and Western blot. The expression of these genes and their products were up-regulated in CB cells treated with 5azaD/TSA. We have also compared the efficacy of an additional HDAC inhibitor valproic acid (VPA) in order to determine its ability to expand HSC ex vivo. VPA was capable of dramatic expansion of CD34+CD90+ cells as well as progenitor cells but was unable to expand SRC. However, unlike the culture exposed to cytokines alone VPA treatment resulted in maintenance of SRC numbers. Currently, we are investigating key candidate genes accountable for the expansion of SRC using a global microarray approach analyzing cells exposed to various chromatin modifying agents in conjunction with their in vivo functional potential. In summary, our data suggest that the loss of SRC can be circumvented by the use of chromatin modifying agents in the culture which results in a slower rate of cell division and is associated with higher expression of a group of HSC regulatory genes.

Id proteins belong to the basic helix-loop-helix family of transcription factors and act as dominant negative forms of E-protein transcriptional activators. Id mediated E protein silencing has an essential role in restricting differentiation and maintaining self-renewal in embryonic stem cells. However, the role of Id1 in adult stem cells including HSCs has not been described thus far. Having detected relatively high levels of Id1 mRNA in murine adult HSCs (compared to the committed myeloid progenitor cells) we examined the in vivo HSC function in Id1 deficient mice. We observed a >2 fold reduction in HSC frequency in the bone marrow in 8-10w old Id1−/− mice compared to Id1+/+ animals, detected by both lin-c-kit+Sca-1+ (LKS) cell surface marker profile and Hoechst 33342 dye efflux - “side population” phenotype, as well as a ~25% decrease in total bone marrow cellularity. Although Id1 deficient HSCs show robust long-term competitive repopulating capacity in primary transplant recipients, they have markedly impaired hematopoietic function upon secondary transplantation. Id1 null HSCs show a higher rate of S-phase entry in vivo as measured by 3 day BrdU incorporation (ko: 79.0±3.9% vs. wt: 49.7±7.4) and faster initial doubling times in response to cytokine stimulation in vitro during the first 2 days of culture. This failure to maintain normal HSC numbers and the diminished repopulating capacity, in the presence of enhanced cell cycling, suggests a defect in the regulation of self-renewal in Id1 deficient HSCs. Considering the general function of Id1 as an inhibitor of differentiation, the observed effect of Id1 loss could be explained by the excessive recruitment of LKS cells into the actively proliferating differentiated progenitor pool, at the expense of their self-renewal capacity. Consistent with this, sorted Id1−/− HSCs show accelerated expression of cell surface lineage markers in vitro and an increased ratio of CFU-S8 /CFU-S12 in the in vivo spleen colony forming assay. Global gene expression profiling of Id1+/+ vs. Id1−/− hematopoietic cells (using Affymetrix MOE430 Plus chips) revealed insignificant transcriptional deregulation in the committed myeloid progenitor subsets (CMP, GMP, MEP) in the absence of Id1. Meanwhile, Id1−/− HSCs showed a marked change in gene expression pattern (more than 1500 genes with a ≥2 fold difference in expression levels). Differentially regulated transcripts in Id1+/+ vs. Id1−/− HSCs significantly overlap (~30%) with the observed changes in gene expression that accompany the transition of HSCs to the common myeloid progenitor phenotype. Specifically, genes such as c/EBPα and GATA1 are significantly upregulated in Id1 null immunophenotypic HSCs, consistent with an earlier than normal commitment to myelo-erythroid differentiation. In contrast, several known transcriptional regulators of HSC self-renewal (Bmi1, Gfi1, HoxB4) show no significant change in expression pattern. These data clearly indicate the unique role of Id1 in regulating HSC self-renewal by restricting the rate of HSC commitment to the myeloid progenitor cell fate.