Showing papers by "University of Florence published in 1992"

Reciprocal regulatory effects of IFN-gamma and IL-4 on the in vitro development of human Th1 and Th2 clones.

Abstract: The effects exerted on the in vitro development of purified protein derivative (PPD)-specific or Dermatophagoides pteronyssinus group I (Der p I)-specific T cell lines (TCL) and T cell clones (TCC) by IL-4 or IFN-gamma addition or neutralization in human PBMC cultures were examined. PBMC from two normal individuals, which were stimulated with PPD and then cultured in IL-2 alone, developed into PPD-specific TCL and TCC able to produce IFN-gamma and IL-2 but not IL-4 and IL-5 (Th1-like). IFN-gamma or anti-IL-4 antibody addition in bulk cultures before cloning did not influence the PPD-specific TCL profile of cytokine production. In contrast, the addition of IL-4 resulted in the development of PPD-specific TCL and TCC able to produce not only IFN-gamma and IL-2 but also IL-4 and IL-5. PBMC from one atopic Der p I-sensitive patient, which were stimulated with Der p I and then cultured in IL-2 alone, developed into Der p I-specific TCL and TCC able to produce IL-5 and large amounts of IL-4 but no IFN-gamma (Th2-like). The addition in bulk cultures, before cloning, of either IFN-gamma or anti-IL-4 antibody markedly inhibited the development of Der p I-specific T cells into IL-4- and IL-5-producing TCL. Accordingly, the development into Der p I-specific Th2-like TCC was significantly reduced by the addition of IFN-gamma in bulk culture and was virtually suppressed by the presence of both IFN-gamma and anti-IL-4 antibody. These data suggest that the presence or the absence of IL-4 and IFN-gamma in bulk cultures of PBMC before cloning may have strong regulatory effects on the in vitro development of human CD4+ T cells into Th1 or Th2 clones.

Genetic evidence for a novel familial Alzheimer's disease locus on chromosome 14

Abstract: Familial Alzheimer's disease (FAD) has been shown to be genetically heterogeneous, with a very small proportion of early onset pedigrees being associated with mutations in the amyloid precursor protein (APP) gene on chromosome 21, and some late onset pedigrees showing associations with markers on chromosome 19. We now provide evidence for a major early onset FAD locus on the long arm of chromosome 14 near the markers D14S43 and D14S53 (multipoint lod score z = 23.4) and suggest that the inheritance of FAD may be more complex than had initially been suspected.

MELAS: Clinical features, biochemistry, and molecular genetics

Abstract: We studied 23 patients with clinically defined mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), 25 oligosymptomatic or asymptomatic maternal relatives, and 50 mitochondrial disease control subjects for the presence of a previously reported heteroplasmic point mutation at nt 3,243 in the transfer RNA(Leu(UUR)) gene of mitochondrial DNA. We found a high concordance between clinical diagnosis of MELAS and transfer RNA(Leu(UUR)) mutation, which was present in 21 of the 23 patients with MELAS, all 11 oligosymptomatic and 12 of 14 asymptomatic relatives, but in only five of 50 patients without MELAS. The proportion of mutant genomes in muscle ranged from 56 to 95% and was significantly higher in the patients with MELAS than in their oligosymptomatic or asymptomatic relatives. In subjects in whom both muscle and blood were studied, the percentage of mutations was significantly lower in blood and was not detected in three of 12 asymptomatic relatives. The activities of complexes I + III, II + III, and IV were decreased in muscle biopsies harboring the mutation, but there was no clear correlation between percentage of mutant mitochondrial DNAs and severity of the biochemical defect.

IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones.

Abstract: The cytolytic potential of a total number of 118 CD4+ human T cell clones specific for purified protein derivative (PPD) from Mycobacterium tuberculosis, tetanus toxoid, Lolium perenne group I allergen (Lol p I), Poa pratensis group IX allergen (Poa p IX), or Toxocara canis excretory/secretory antigen(s) (TES) was assessed by both a lectin (PHA)-dependent and a MHC-restricted lytic assay and compared with their profile of cytokine secretion. The majority of clones with Th1 or Th0 cytokine profile exhibited cytolytic activity in both assays, whereas Th2 clones usually did not. There was an association between the cytolytic potential of T cell clones and their ability to produce IFN-gamma, even though IFN-gamma produced by T cell clones was not responsible for their cytolytic activity. IL-4 added in bulk culture before cloning inhibited not only the differentiation of PPD-specific T cells into Th1-like cell lines and clones, but also the development of their cytolytic potential. The depressive effect of IL-4 on the development of PPD-specific T cell lines with both Th1 cytokine profile and cytolytic potential was dependent on early addition of IL-4 in bulk cultures. In contrast, the addition in bulk culture of IFN-gamma enhanced both the cytolytic activity of PPD-specific T cell lines, as well as the proportion of PPD-specific T cell clones with cytolytic activity. The addition in bulk cultures before cloning of IFN-gamma or IFN-alpha favored the development of TES-specific and Poa p IX-specific T cells into T cell clones showing a Th0 or even a Th1, rather than a Th2, cytokine profile. Accordingly, most of TES- and Poa p IX-specific T cell clones derived from cultures containing IFN-gamma or IFN-alpha displayed strong cytolytic activity. These data indicate that the majority of human T cell clones that produce IFN-gamma, but not IL-4 (Th1-like), as well as of T cell clones that produce IFN-gamma in combination with IL-4 (Th0-like) are cytolytic. More importantly, they demonstrate that the addition of IFN (alpha and gamma) or IL-4 in bulk cultures before cloning may influence not only the cytokine profile of human CD4+ T cell clones but also their cytolytic potential.

Human TH1 and TH2 subsets : regulation of differentiation and role in protection and immunopathology

Abstract: A large body of evidence has accumulated suggesting the existence of human TH1 and TH2 subsets, reminiscent of those described for mouse T cells. Human TH1 cells develop in response to intracellular b

Faster superoxide dismutase mutants designed by enhancing electrostatic guidance

Abstract: THE enzyme Cu, Zn superoxide dismutase (SOD) protects against oxidative damage by dismuting the superoxide radical O.−2 to molecular oxygen and hydrogen peroxide1–3 at the active-site Cuion4,5 in a reaction that is rate-limited by diffusion3,6 and enhanced by electrostatic guidance7–10. SOD has evolved to be one of the fastest enzymes known ( Vmax ~ 2 x 109 M−1 s−1)6,11. The new crystal structures of human SOD12 show that amino-acid site chains that are implicated in electrostatic guidance8 (Glu 132, Glu 133 and Lys 136) form a hydrogen-bonding network. Here we show that site-specific mutants that increase local positive charge while maintaining this orienting network (Glu→Gin) have faster reaction rates and increased ionic-strength dependence, matching brownian dynamics simulations incorporating electrostatic terms. Increased positive charge alone is insufficient: one charge reversal (Glu→Lys) mutant is slower than the equivalent charge neutralization (Glu→Gin) mutant, showing that the newly introduced positive charge disrupts the orienting network. Thus, electrostatically facilitated diffusion rates can be increased by design, provided the detailed structural integrity of the active-site electrostatic network is maintained.

Pharmacokinetic Drug Interactions of Macrolides

Abstract: The macrolide antibiotics include natural members, prodrugs and semisynthetic derivatives. These drugs are indicated in a variety of infections and are often combined with other drug therapies, thus creating the potential for pharmacokinetic interactions. Macrolides can both inhibit drug metabolism in the liver by complex formation and inactivation of microsomal drug oxidising enzymes and also interfere with microorganisms of the enteric flora through their antibiotic effects. Over the past 20 years, a number of reports have incriminated macrolides as a potential source of clinically severe drug interactions. However, differences have been found between the various macrolides in this regard and not all macrolides are responsible for drug interactions. With the recent advent of many semisynthetic macrolide antibiotics it is now evident that they may be classified into 3 different groups in causing drug interactions. The first group (e.g. troleandomycin, erythromycins) are those prone to forming nitrosoalkanes and the consequent formation of inactive cytochrome P450-metabolite complexes. The second group (e.g. josamycin, flurithromycin, roxithromycin, clarithromycin, miocamycin and midecamycin) form complexes to a lesser extent and rarely produce drug interactions. The last group (e.g. spiramycin, rokitamycin, dirithromycin and azithromycin) do not inactivate cytochrome P450 and are unable to modify the pharmacokinetics of other compounds. It appears that 2 structural factors are important for a macrolide antibiotic to lead to the induction of cytochrome P450 and the formation in vivo or in vitro of an inhibitory cytochrome P450-iron-nitrosoalkane metabolite complex: the presence in the macrolide molecules of a non-hindered readily accessible N-dimethylamino group and the hydrophobic character of the drug. Troleandomycin ranks first as a potent inhibitor of microsomal liver enzymes, causing a significant decrease of the metabolism of methylprednisolone, theophylline, carbamazepine, phenazone (antipyrine) and triazolam. Troleandomycin can cause ergotism in patients receiving ergot alkaloids and cholestatic jaundice in those taking oral contraceptives. Erythromycin and its different prodrugs appear to be less potent inhibitors of drug metabolism. Case reports and controlled studies have, however, shown that erythromycins may interact with theophylline, carbamazepine, methylprednisolone, warfarin, cyclosporin, triazolam, midazolam, alfentanil, disopyramide and bromocriptine, decreasing drug clearance. The bioavailability of digoxin appears also to be increased by erythromycin in patients excreting high amounts of reduced digoxin metabolites, probably due to destruction of enteric flora responsible for the formation of these compounds. These incriminated macrolide antibiotics should not be administered concomitantly with other drugs known to be affected metabolically by them, or at the very least, combined administration should be carried out only with careful patient monitoring.(ABSTRACT TRUNCATED AT 400 WORDS)

Fat-storing cells as liver-specific pericytes. Spatial dynamics of agonist-stimulated intracellular calcium transients.

Abstract: Liver perisinusoidal fat-storing cells (FSC) show morphological and ultrastructural characteristics similar to pericytes regulating local blood flow in other organs. In the present study we have analyzed whether FSC respond to local vasoconstrictors such as thrombin, angiotensin-II, and endothelin-1 with an increase in intracellular free calcium concentration ([Ca2+]i) coupled with effective cell contraction. All agonists tested induced a rapid and dose-dependent increase in [Ca2+]i followed by a sustained phase lasting several minutes in confluent monolayers of Fura-2-loaded human FSC. Pharmacological studies performed using different Ca2+ channel blockers indicated that, at least for thrombin and angiotensin-II, the sustained phase is due to the opening of voltage-sensitive membrane Ca2+ channels. To analyze the temporal and spatial dynamics of Ca2+ release in response to these agonists, we performed experiments on individual Fura-2-loaded human FSC using a dual wavelength, radiometric video imaging system. The rise in [Ca2+]i was exclusively localized to the cytoplasm, particularly in the branching processes. Increases in [Ca2+]i more than four-fold were associated with a simultaneous and transient reduction of cell area indicating reversible cell contraction. Our results indicate that the Ca(2+)-dependent contraction of human FSC in vitro may reflect a potential role in regulating sinusoidal blood flow in vivo.

Heart rate variability during the acute phase of myocardial infarction.

Abstract: BACKGROUNDAfter acute myocardial infarction (AMI), several abnormalities of the autonomic control to the heart have been described. Heart rate (HR) variability has been used to explore the neural control to the heart. A low HR variability count measured 7-13 days after AMI is significantly related to a poor outcome. Little information is available on HR variability early after AMI and its relation to clinical and hemodynamic data.METHODS AND RESULTSWe studied 54 consecutive patients (42 men and 12 women; mean age, 60.4 +/- 11 years) with evidence of AMI by collecting the 24-hour HR SD from Holter tapes recorded on day 2 or 3. We also measured HR variability in 15 patients with unstable angina and in 35 age-matched normal subjects. HR variability was lower in AMI than in unstable angina patients (57.6 +/- 21.3 versus 92 +/- 19 msec; p less than 0.001) and controls (105 +/- 12 msec; p less than 0.001). Also, HR variability was greater in non-Q-wave than in Q-wave AMI (p less than 0.0001) and in recombinant ...

Human Th1 and Th2 lymphocytes: their role in the pathophysiology of atopy

Abstract: In human beings, as in mice, two distinct patterns of cytokine secretion have been defined among CD4+ helper T-cell clones. Human type 1 helper (Th1), but not type 2 helper (Th2), cells produce interleukin-2 (IL-2), gamma-interferon (IFN-gamma), and tumor necrosis factor-beta, whereas Th2, but not Th1, cells secrete IL-4 and IL-5, but not IL-2 or IFN-gamma. Other cytokines, such as IL-3, IL-6, GM-CSF, or TNF-alpha, are produced by both Th1 and Th2 cells. Th0 cells, a third Th subset, show combined production of Th1- and Th2-type cytokines. The different cytokine patterns are associated with different functions. In general, Th2 cells provide an excellent helper function for B-cell antibody production, particularly of the IgE class. On the other hand, Th1 cells are responsible for delayed type hypersensitivity reactions and are cytolytic for autologous antigen-presenting cells, including B cells. Most allergen- or helminth-antigen-specific human CD4+ T-cell clones exhibit a Th2 phenotype, whereas most clones specific for bacterial antigens show a Th1 profile. Allergen-specific Th2 cells seem to play a crucial role in atopy. These cells induce IgE production via IL-4 and favor the proliferation, differentiation, and activation of eosinophils via IL-5. In addition, Th2-derived IL-3 and IL-4 are mast-cell growth factors that act in synergy, at least in vitro. Recent evidence indicates that allergen-specific Th2 cells are selectively enriched in tissues affected by allergic inflammation, such as the bronchial mucosa of subjects with allergic asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

Nickel toxicity and peroxidase activity in seedlings of Triticum aestivum L.

Abstract: Ni2+ toxicity was evaluated in Triticum aestivum L. by its effects on root and shoot length, dry matter production and water content. Over a threshold value of 20 mmol m−3 Ni2+ the degree of toxicity increases as a function of the Ni2+ concentration in the medium. Ni2+-treated roots show enhanced lipid peroxidation; the higher Ni2+ treatment (40mmol m−3) also increases leakage of K+. In roots and shoots, Ni2+ enhances both guaiacol and syringaldazine extracellular peroxidase activity. The increase in extracellular peroxidase activity is also associated with an increase in the phenolic contents of roots and shoots. The observed growth inhibition might be partly the result of the effect of Ni2+ on cell turgor and cell-wall extensibility. Intracellular soluble peroxidases are also stimulated by Ni2+; such effects, independently of the substrate, were detected in extracts of Ni2+-treated shoots at a lower Ni2+ concentration than in the roots. Intracellular peroxidases might act as scavengers of peroxide radicals produced as a result of nickel toxicity.

Type 1 T helper and type 2 T helper cells: Functions, regulation and role in protection and disease

Abstract: Two very distinct cytokine secretion patterns have been defined among murine CD4+ T cells. Type 1 helper (TH1), but not type 2 helper (TH2), cells produce interleukin (IL)-2, gamma-interferon (IFN-gamma) and tumour necrosis factor-beta, whereas TH2, but not TH1, cells express IL-4, IL-5, IL-6 and IL-10. The different cytokine patterns lead to different functions of the two types of T cell. In general, TH2 cells are excellent helpers for B-cell antibody secretion, particularly IgE responses. On the other hand TH1 cells induce delayed-type hypersensitivity reactions. There is general agreement that the different functional subsets of TH cells arise post-thymically from a common pool of precursors and as a consequence of activation of antigen. However, the factors affecting differentiation of TH precursors into the TH1 or TH2 subsets are still unclear. Mutual cross-regulation between TH1 (via IFN-gamma) and TH2 (via IL-10) has also been reported. Recently, human T cell clones similar to murine TH1 and TH2 cells have been demonstrated. Most allergen- or helminthic antigen-specific CD4+ human T cell clones have a TH2 phenotype, whereas the majority of T-cell clones specific for mycobacterial antigens or antigens responsible for type IV hypersensitivity exhibit a TH1 phenotype. Human TH2 clones provide B-cell help for IgE synthesis, whereas most TH1 clones are cytolytic for antigen-presenting cells, including B lymphocytes. It is highly probable that the selective or preferential activation of CD4+ T-cell subsets secreting defined patterns of cytokines is of major importance in determining the class of immune effector function, thus influencing both protection and immunopathology.

Transient intermittent lymphocyte activation is responsible for the instability of angina.

Abstract: BACKGROUNDBlood clotting activation is an important component of the inflammatory response; the outbursts of unstable angina are usually associated with increased thrombin formation and coronary mural thrombosis.METHODS AND RESULTSTo investigate 1) whether monocyte activation is responsible for the enhanced thrombin formation during bursts of unstable angina and 2) what mechanism(s) might be responsible for monocyte activation, we studied patients with unstable angina (n = 31), stable effort angina (n = 23), left endoventricular thrombosis (n = 8), and control subjects (n = 44), measuring plasma fibrinopeptide A (FPA) levels and the capacity of monocytes to express procoagulant activity (PCA) and of lymphocytes to modulate this expression. Patients with unstable angina and patients with endoventricular thrombosis had significantly (p less than 0.0001) higher FPA plasma levels than patients with effort angina and control subjects. However, only monocytes from unstable angina patients expressed significantl...

From open ponds to vertical alveolar panels: the Italian experience in the development of reactors for the mass cultivation of phototrophic microorganisms

Abstract: The need to develop new concepts in reactor design and the growing interest inSpirulina prompted our group to abandon open ponds in the seventies and to focus interest mainly on closed systems. Two substantially different closed photobioreactors have been developed and are at present under investigation in our Research Centre: the tubular photobioreactor (made of rigid or collapsible tubes) and the recently devised vertical alveolar panel (VAP) made of 1.6-cm-thick Plexiglas alveolar sheets.

Long-term study of anterior cruciate ligament reconstruction for chronic instability using the central one-third patellar tendon and a lateral extraarticular tenodesis

Abstract: Forty-four patients with symptomatic chronic anterior cruciate ligament instabilities that had been recon structed with the central one-third patellar tendon and a lateral extraarticular iliotibial band tenodesis were studied at an average followup of 7 years (range, 4 to 10). The cases with associated medial, lateral, or pos terior laxity were not included, nor were the cases with more than minimal preoperative degenerative changes. The average age at surgery was 21 years (range, 16 to 33). A postoperative cast was used for 4 weeks.Satisfactory objective stability, which was defined as a KT-1000 side-to-side difference of up to 5 mm at the manual maximum test, was obtained in 37 (84%) of the patients. In 25 patients (57%), stability was restored within normal limits (≤3 mm). No deterioration of the KT-1000 stability was noted at two follow-up visits performed by the same examiner (at an average of 4 and 7 years). A return to high-risk sports was possible in 27 (62%) of the patients.Difficulties in regain...

Lupus anticoagulant and anticardiolipin antibodies in young adults with cerebral ischemia.

Abstract: Our study evaluates in an unselected young population with cerebral ischemia the frequency of antiphospholipid antibodies; the relationship of antiphospholipid antibodies to conventional risk factors for and pathological mechanisms of cerebral ischemia; and the risk of recurrence of cerebral ischemia or systemic thrombotic events in patients with antiphospholipid antibodies compared with those without. We prospectively tested for antiphospholipid antibodies in 55 of 59 young (aged 15-44 years) adults consecutively examined for ischemic stroke (n = 44) or transient ischemic attack (n = 11). These patients underwent a complete clinical and laboratory assessment for cerebral ischemia and had a 3-year mean follow-up. Ten patients (18%), all with stroke, had antiphospholipid antibodies. Antiphospholipid antibodies were significantly more frequent in women than in men (Fisher's test, p = 0.014). Two patients with antiphospholipid antibodies had a new diagnosis of systemic lupus erythematosus. On angiography, none of the patients with antiphospholipid antibodies had extracranial lesions. Patients with antiphospholipid antibodies had significantly more prior cerebral events (Fisher's test, p = 0.014), and, by survival analysis, higher probability of cerebral ischemic or systemic thrombotic events during follow-up than patients without (log rank test, p less than 0.005). We conclude that the prevalence of antiphospholipid antibodies is rather high in young adults with cerebral ischemia; that patients with cerebral ischemia and antiphospholipid antibodies may have unrecognized systemic lupus erythematosus; and that, among young patients with cerebral ischemia, patients with antiphospholipid antibodies constitute a subgroup at high risk of cerebral ischemic or systemic thrombotic recurrence. Prevention in this latter group may require close follow-up and treatment.

Rapid regeneration of the actin-myosin power stroke in contracting muscle.

Abstract: AT the molecular level, muscle contraction is the result of cyclic interaction between myosin crossbridges, which extend from the thick filament, and the thin filament, which consists mainly of actin. The energy for work done by a single crossbridge during a cycle of attachment, generation of force, shortening and detachment is believed to be coupled to the hydrolysis of one molecule of ATP1,2. The distance the actin filament slides relative to the myosin filament in one crossbridge cycle has been estimated as 12 nm by step-length perturbation studies on single fibres from frog muscle3,4. The 'mechanical' power stroke of the attached crossbridge can therefore be defined as 12-nm shortening with a force profile like that shown by the quick recovery of force following a length perturbation. According to this definition, power strokes cannot be repeated faster than the overall ATPase rate. Here, however, we show that the power stroke can be regenerated much faster than expected from the ATPase rate. This contradiction can be resolved if, in the shortening muscle, the free energy of ATP hydrolysis is used in several actin–myosin interactions consisting of elementary power strokes each of 5–10 nm.

Myosin head movements are synchronous with the elementary force-generating process in muscle.

Abstract: Motor proteins such as myosin, dynein and kinesin use the free energy of ATP hydrolysis to produce force or motion, but despite recent progress their molecular mechanism is unknown. The best characterized system is the myosin motor which moves actin filaments in muscle. When an active muscle fibre is rapidly shortened the force first decreases, then partially recovers over the next few milliseconds. This elementary force-generating process is thought to be due to a structural 'working stroke' in the myosin head domain, although structural studies have not provided definitive support for this. X-ray diffraction has shown that shortening steps produce a large decrease in the intensity of the 14.5 nm reflection arising from the axial repeat of the myosin heads along the filaments. This was interpreted as a structural change at the end of the working stroke, but the techniques then available did not allow temporal resolution of the elementary force-generating process itself. Using improved measurement techniques, we show here that myosin heads move by about 10 nm with the same time course as the elementary force-generating process.

Human parvovirus B19 infection in hemophiliacs first infused with two high-purity, virally attenuated factor VIII concentrates.

Abstract: Human parvovirus B19 can be transmitted by coagulation factor concentrates and is highly resistant to virucidal methods. To evaluate whether the additional removal of virus by chromatographic methods during the manufacture of high-purity concentrates reduces the risk of B19 transmission, we have prospectively evaluated the rate of anti-B19 seroconversion in two groups of susceptible (anti-B19 negative) hemophiliacs infused with high-purity, heated (pasteurized) or solvent-detergent-treated factor VIII concentrates. Both products infected a relatively high proportion of patients (nine of 20).