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Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient’s keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

Abstract Objective--To assess the prognostic value of stress echocardiography as an adjunct to exercise electrocardiography in patients with uncomplicated acute myocardial infarction. Design--496 patients underwent a maximum exercise EGG and [...]

Objective:To investigate the effectiveness of an innovative electrical stimulation (ES) therapy as adjuvant treatment for chronic wounds of various aetiology, in terms of pain and ulcer healing.Method:Patients with chronic limb ulcers were enrolled for the study and randomised into the intervention or control group. The intervention group received conventional treatment plus ES therapy (FREMS; Lorenz Lifetech) while the control group received only conventional treatment. Each ES treatment cycle consisted of 12 sessions performed in 4 weeks (three sessions/week). All patients were treated until full wound healing occurred, or for a maximum of 9 ES cycles, with a 2-week rest between each cycle.Results:A total of 60 patients were enrolled in the study and randomised into the two groups: the intervention group (n=30) and the control group (n=30). During follow-up, some patients terminated the protocol because they reached the ulcer closure before the maximum of 9 cycles. The analysis of the effect of ES on pain and ulcer healing was performed on all patients who underwent at least two consecutive clinical evaluations (two cycles), in order to reach a compatible sample size with the primary objective (one patient withdrew). In both groups, there was a significant reduction of pain compared with baseline (p < 0.05), starting from T6visit in the first cycle. In particular, there was a significant reduction of pain in the intervention group compared with the control group after 14 days, and this reduction continued until the end of the second cycle. Similarly, there was a significant reduction of PUSH tool score in the intervention group compared with the control group after 14 days, and this reduction continued until the end of the second cycle.Conclusion:Data collected in this study support data in the literature. Analysis of longitudinal data analysed by simple models and complex models suggest that the ES therapy had a positive and significant effect on pain reduction (VAS) and on the improvement of ulcer healing process in terms of the PUSH tool total index compared with conventional treatment, and may have induced a significant acceleration of the wound-healing process.Declaration of interest:There were no external sources of funding for this trial. The authors have no financial, commercial or social conflicts of interest to declare regarding the article or its content.

Natural toxins are the product of a long-term evolution, and act on essential mechanisms in the most crucial and vital processes of living organisms. They can attack components of the protein synthesis machinery, actin polymerization, signal transduction pathways, intracellular trafficking of vesicles as well as immune and inflammatory responses. For this reason, toxins have increasingly being used as valuable tools for analysis of cellular physiology, and in the recent years, some of them are used medicinally for the treatment of human diseases. This review is devoted to protein toxins of bacterial origin, specifically those toxins that are currently used in therapy or those under study for their potential clinical applications. Bacterial protein toxins are all characterized by a specific mechanism of action that involves the central molecular pathways in the eukaryotic cell. Knowledge of their properties could be used for medical purposes.

In the striatum, dopamine D1R (D1 receptor) activation potentiates NMDA (N-methyl-D-aspartate) transmission and is required for NMDA-mediated long-term potentiation at corticostriatal synapses. By using a combination of co-immunoprecipitation, pull-out with glutathione S-transferase-fusion proteins and bioluminescence resonance energy transfer, we have reported that the D1R forms a heteromeric complex with the NMDAR (NMDA receptor) and that this mechanism is crucial to recruit the D1R to the postsynaptic density. By using confocal and radioligand-binding assay, we also demonstrated that the interaction with NMDAR abolishes agonist-mediated D1R sequestration, indicating that oligomerization with NMDAR could represent a novel regulatory mechanism modulating D1R cellular trafficking and desensitization.

The authors carried out a 1-year, randomized, placebo-controlled trial of idebenone in 29 patients with Friedreich ataxia. They found significant reductions of interventricular septal thickness and left ventricular mass in the idebenone group vs the placebo group, with no improvement in other heart ultrasound measures or neurologic condition. The absolute cardiac changes were modest, but the findings suggest that larger trials should assess whether idebenone reduces ventricular hypertrophy in patients with Friedreich ataxia.

OBJECTIVE: Nerve growth factor (NGF) has antiproliferative and differentiating effects in neuroendocrine tumors. In cell lines derived from small cell lung cancer (SCLC), NGF treatment stimulates NGF receptor expression, activates NGF secretion, inhibits proliferation and abrogates invasion. Since these effects are lost upon NGF withdrawal, it is relevant to identify other differentiation factors that may co-operate with the NGF system to control SCLC growth and differentiation. DESIGN: Retinoic acid (RA), which has been shown to inhibit cell transformation and proliferation, modulates the expression of NGF receptors and the sensitivity to NGF in different cell models. In the present study, we have investigated whether NGF and RA may interact to control the proliferation of SCLC cell lines. METHODS: SCLC cells were exposed to 50 ng/ml NGF or 1 microM all-trans RA for different times. Cell proliferation was measured by the [(3)H]thymidine incorporation test and NGF receptor expression was evaluated by immunofluorescence. RESULTS: We found that RA increased the expression of both trkA and p75 NGF receptors in NCI-N-592 and GLC8 cell lines and prevented the loss of both NGF production and NGF receptor expression occurring when NGF treatment was discontinued. As a result, RA, which did not inhibit the proliferation of untreated cells, abolished NGF withdrawal-related increase in cell proliferation both in vitro and in vivo, thus making permanent the antiproliferative effects of NGF. CONCLUSIONS: These data suggest that combined treatments with NGF and RA or mimicking drugs may represent a strategy to be further investigated for the treatment of SCLC.

Vibrio parahaemolyticus is a marine bacterium known to be a common cause of seafood gastroenteritis worldwide. The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of V. parahaemolyticus. TDH causes intestinal fluid secretion as well as cytotoxicity in a variety of cell types. In this study, we investigated the interplay between the hemolysin's enterotoxic and cytotoxic effects by using both human and rat cell monolayers. As revealed by microspectrofluorimetry, the toxin causes a dose-dependent increase in intracellular free calcium in both Caco-2 and IEC-6 cells. This effect was reversible only when low toxin concentrations were tested. The TDH-activated ion influx pathway is not selective for calcium but admits ions such sodium and manganese as well. Furthermore, in the same range of concentration, the hemolysin triggers a calcium-dependent chloride secretion. At high concentrations, TDH induces a dose-dependent but calcium-independent cell death as assessed by functional, biochemical, and morphological assays.

Toxins A and B from the anaerobic bacterium Clostridium difficileare the causative agents of the antibiotic‐associated pseudomembraneous colitis. At the subcellular level, they inhibit the Rho family GTPases, thus causing alterations of the actin cytoskeleton. The cytoskeletal integrity is also controlled by the redox state of cells. Therefore, we have evaluated whether an oxidative imbalance could be involved in the toxin‐induced cytopathic effects. Our results indicate that both toxins induce oxidative stress with a significant depletion of protein SH‐groups. These responses and the cytoskeleton‐dependent cell retraction and rounding are significantly counteracted by N‐acetylcysteine but not by α‐tocopherol. Our study provides the first evidence that the thiol supplier N‐acetylcysteine impairs the cellular intoxication by acting on the cytoskeleton integrity. This also suggests a possible beneficial role for this drug during therapeutic intervention.

Vibrio parahaemolyticus is a marine bacterium known to be the leading cause of seafood gastroenteritis worldwide. A 46-kDa homodimer protein secreted by this microorganism, the thermostable direct hemolysin (TDH), is considered a major virulence factor involved in bacterial pathogenesis since a high percentage of strains of clinical origin are positive for TDH production. TDH is a pore-forming toxin, and its most extensively studied effect is the ability to cause hemolysis of erythrocytes from different mammalian species. Moreover, TDH induces in a variety of cells cytotoxic effects consisting mainly of cell degeneration which often leads to loss of viability. In this work, we examined the cellular changes induced by TDH in monolayers of IEC-6 cells (derived from the rat crypt small intestine), which represent a useful cell model for studying toxins from enteric bacteria. In experimental conditions allowing cell survival, TDH induces a rapid transient increase in intracellular calcium as well as a significant though reversible decreased rate of progression through the cell cycle. The morphological changes seem to be dependent on the organization of the microtubular network, which appears to be the preferential cytoskeletal element involved in the cellular response to the toxin.

A case is described of a 54 year old woman who had acute pericarditis with large exudative effusion accompanied by severe right and left ventricular failure. The patient was finally diagnosed with carcinoid heart disease from an ovarian carcinoid teratoma. She was treated with octreotide--a somatostatin analogue--followed by radical surgical resection of the neoplasm. At one year follow up only mild carcinoid tricuspid regurgitation remained. Only 16 cases of carcinoid heart disease from an ovarian primary have been described in literature. Moreover clinically manifest acute, non-metastatic pericarditis and left heart failure are not considered as possible presentations of carcinoid heart disease, whatever the origin. In a recent series a small pericardial effusion was considered an infrequent and unexpected echocardiographic finding in carcinoid heart patients. One case of "carcinoid pericarditis" has previously been described as a consequence of pericardial metastasis. Left sided heart involvement is usually caused by bronchial carcinoids or patency of foramen ovale; both were excluded in the case presented.

Objective: To evaluate the pathogenicity of a recently discovered arthropod-transmitted bunyavirus (Toscana virus) on the CNS in children and to provide information on the epidemiologic and clinical aspects of Toscana virus infection. Study design: Case-series analysis of children hospitalized with clinical and cerebrospinal fluid examination compatible with a CNS disease of viral origin. Methods: Cerebrospinal fluid, acute, and convalescent sera were investigated for conventional neurotropic viruses and for Toscana and tickborne encephalitis viruses. A clinical-epidemiologic analysis was carried out on confirmed Toscana virus cases to clarify the profile of Toscana virus infection in children. Results: The study indicates that (1) Toscana virus has been endemic in the Siena province for at least 15 years; (2) the virus is responsible for at least 80% of acute viral infections of the CNS in children throughout the summertime; (3) the clinical signs and symptoms range from aseptic meningitis to meningoencephalitis; (4) infected children resided habitually or temporarily in rural or suburban areas of the Siena province, where ecological characteristics allow arthropods to be peridomestic in human settlements. Conclusions: Toscana virus is the most common viral agent involved in acute infections of CNS in children in central Italy. (J Pediatr 1998;132:144-8)

Cytotoxic necrotizing factor 1 (CNF1), a 110-kDa protein toxin from pathogenic Escherichia coli induces actin reorganization into stress fibers and retraction fibers in human epithelial cultured cells allowing them to spread. CNF1 is acting in the cytosol since microinjection of the toxin into HEp-2 cells mimics the effects of the externally applied CNF1. Incubation in vitro of CNF1 with recombinant small GTPases induces a modification of Rho (but not of Rac, Cdc42, Ras, or Rab6) as demonstrated by a discrete increase in the apparent molecular weight of the molecule. Preincubation of cells with CNF1 impairs the cytotoxic effects of Clostridium difficile toxin B, which inactivates Rho but not those of Clostridium sordellii LT toxin, which inhibits Ras and Rac. As shown for Rho-GTP, CNF1 activates, in a time- and dose-dependent manner, a cytoskeleton-associated phosphatidylinositol 4-phosphate 5-kinase. However, neither the phosphatidylinositol 4,5-bisphosphate (PIP2) nor the phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) or 3,4,5-trisphosphate (PIP3) cellular content were found increased in CNF1 treated HEp-2 cells. Cellular effects of CNF1 were not blocked by LY294002, a stable inhibitor of the phosphoinositide 3-kinase. Incubation of HEp-2 cells with CNF1 induces relocalization of myosin 2 in stress fibers but not in retraction fibers. Altogether, our data indicate that CNF1 is a toxin that selectively activates the Rho GTP-binding protein, thus inducing contractility and cell spreading.

A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor. While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.

Certain strains of the anaerobic bacterium Bacteroides fragilis are known to produce an enterotoxin of about 20 kDa which is able to induce a fluid response in ligated intestinal loops and a cytotoxic response in HT-29 cells. It presents protease activity, belonging to a family of metalloproteases termed metzincins. In order to investigate the mode of action of the enterotoxin in cultured cells, we performed a study with HT-29 cells, using both fluoresence and electron microscopy. Treated cells underwent morphological changes, mainly consisting of the retraction of the cell body and the formation of numerous blebs on the cell surface. The microfilament system was reorganized, the F-actin being condensed as a ring at the cell periphery, whereas other cell organelles appeared to be unaffected. All these changes, clearly visible after 3 h of exposure to the toxin, were reversed within 24 h of treatment. By inhibiting the protease activity of the toxin with specific metal chelators, the cytoskeletal effects were also prevented. Thus, B. fragilis enterotoxin appears to act on cells by reversibly modifying the actin cytoskeleton, an effect probably dependent on its proteolytic activity.

Experiments done on in vitro-cultured cells exposed to toxin A from C. difficile showed a series of cytopathologic changes leading to cell retraction and rounding accompanied by the marginalization of the nucleus, which localized at one pole of the cell. Cytoskeleton appeared to be strongly involved in such modifications. In particular, the microfilament system seemed to be involved in cell retraction, while microtubule network integrity and function seemed to be necessary for the nuclear displacement. The carboxylic ionophore monensin completely blocked the cytopathic effect when added with the toxin. The serine protease inhibitor chymostatin appeared to be protective also upon addition long after the end of the binding step. The Ca2(+)-dependent cytosolic protease inhibitors antipain and leupeptin were uneffective in protecting cells. Thus, our results suggest the involvement of an acidic compartment and the action of a serine protease in toxin A-induced cytopathic effect.

In central Italy, acute lymphocytic meningitis and meningoencephalitis due to a Phlebotomus transmitted virus (Phlebovirus Toscana, TOSv) occurring throughout the summer are frequently observed. Several serum specimens of patients hospitalized with a clinical picture of viral meningitis/meningoencephalitis showed anti TOS-IgG reactivity suggestive of a previous infection occurring at an unknown time in the past. This observation led us to design a serological investigation of 83 household contacts of 46 summertime CNS infection patients (index cases) with the purpose of evaluating the percentage of both IgG and IgM in seropositive healthy individuals, living, like the index cases, in areas at high risk of phlebotomine sandfly bites. The serological study was carried out using an ELISA method: 22% of the sera showed a reactivity for anti-TOS IgG antibodies and 6% resulted IgG/IgM positive; none of them reported having had any symptoms of CNS involvement; the distribution of seropositive cases was similar in contacts of both TOS-confirmed and TOS-negative cases. Our results indicate: (1) that phlebovirus Toscana is frequently implicated in cases with CNS disease as well as in infections occurring without neurological involvement; (2) in our country the ecological requirements encompass the conditions in and around the human settlements for phlebotomine sandflies to become peridomestic thus amplifying the risk of TOSv infections, which are in fact widespread and frequent in Siena and its surroundings.

Toxigenic strains of the anaerobic bacterium Clostridium difficile produce at least two large, single-chain protein exotoxins involved in the pathogenesis of antibiotic-associated diarrhea and colitis. Toxin A (CdA) is a cytotoxic enterotoxin, while toxin B (CdB) is a more potent cytotoxin lacking enterotoxic activity. This study dealt with CdB, providing the first evidence that intestinal cells exposed to this toxin exhibit typical features of apoptosis in that a significant proportion of the treated cells displayed nuclear fragmentation and chromatin condensation. In keeping with ultrastructural data, CdB-treated cells showed the typical flow cytometric hallmark of apoptosis consisting of a distinct sub-G1 peak. The CdB-induced apoptotic response was dose and time dependent and not simply due to the actin-disrupting effect of the toxin or to the subsequent impairment of cell anchorage. Rather, the inhibition of proteins belonging to the Rho family due to CdB seems to play a role in the induction of apoptosis in intestinal cells. The origin of cells and the growth rate may also be cofactors relevant to such a response.

Cytotoxic necrotizing factor type 1 (CNF1), a 110-kDa toxin-like protein from pathogenic Escherichia coli strains, induces an actin cytoskeleton reorganization consisting of the formation of prominent stress fibers by permanent activation of the small GTP-binding protein Rho. Since p21Rho regulates tight-junction permeability and perijunctional actin reorganization in epithelial intestinal cells (A. Nusrat, M. Giry, J. R. Turner, S. P. Colgan, C. A. Parkos, E. Lemichez, P. Boquet, and J. L. Madara, Proc. Natl. Acad. Sci. USA 92:10629-10633, 1995), we used polarized T84 epithelial intestinal cell monolayers to examine whether CNF1 could affect microvillus structure, transepithelial resistance, and polymorphonuclear leukocyte (PMN) transmigration. Incubation of T84 cells with CNF1 did not influence transepithelial resistance, suggesting that barrier function and surface polarity were not affected by the toxin. However, CNF1 effaced intestinal cell microvilli and induced a strong decrease of PMN transepithelial migration in either the luminal-to-basolateral or the basolateral-to-luminal direction. CNF1 could thus be a virulence factor exhibiting a new type of combined activity consisting of effacing of microvilli and occlusion of the epithelial barrier to PMNs. Attenuated transepithelial migration of PMNs could result in the enhanced growth and protection of luminal bacteria.

Cytotoxic necrotizing factor type 1 (CNF1) induces in HEp-2 cells an increase in F-actin structures, which was detectable by fluorescence-activated cell sorter analysis 24 h after addition of this factor to the culture medium. Increase in F-actin was correlated with the augmentation of both the cell volume and the total cell actin content. Actin assembly-disassembly is controlled by small GTP-binding proteins of the Rho family, which have been reported recently to be modified by CNF1 treatment. Clostridium difficile toxin B and Clostridium botulinum exoenzyme C3, both known to act on the Rho GTPase, were used as biological tools to study the effect of CNF1 on this protein. CNF1 incubated before, during, or after exposure to the chimeric toxin C3B (which is the product of a genetic fusion between the DNA coding for C3 and the one coding for the B fragment of diphtheria toxin) protected HEp-2 cells from the disruption of F-actin structures caused by inactivation of the Rho GTPase through its ADP-ribosylation. On the other hand, C. difficile toxin B cytopathic effect was not observed upon preincubation of cells with CNF1. Toxins acting through a Rho-independent mechanism, such as cytochalasin D and Clostridium spiroforme iota-like toxin, could not be modified in their cellular activities by CNF1 treatment. All of our results suggest that CNF1 modifies the Rho molecule, thus probably protecting this GTPase from further bacterial toxin modification.

In rat intestinal crypt cells, Clostridium difficile toxin A induces (i) early cytoskeletal alterations involving the whole population and (ii) late effects in 30 to 40% of the cells, consisting mainly of surface blebbing and nuclear fragmentation. All these effects were Ca2+ independent and were not abolished by protein synthesis inhibitors.

Cytotoxic necrotizing factor type 2 (CNF2) produced by Escherichia coli strains isolated from intestinal and extraintestinal infections is a dermonecrotic toxin of 110 kDa. We cloned the CNF2 gene from a large plasmid carried by an Escherichia coli strain isolated from a lamb with septicemia. Hydropathy analysis of the deduced amino acid sequence revealed a largely hydrophilic protein with two potential hydrophobic transmembrane domains. The N-terminal half of CNF2 showed striking homology (27% identity and 80% conserved residues) to the N-terminal portion of Pasteurella multocida toxin. Methylamine protection experiments and immunofluorescence studies suggested that CNF2 enters the cytosol of the target cell through an acidic compartment and induces the reorganization of actin into stress fibers. Since the formation of stress fibers in eukaryotic cells involves Rho proteins, we radiolabeled these small GTP-binding proteins from CNF2-treated and control cells with a Rho-specific ADP-ribosyltransferase. The [32P]ADP-ribosylated Rho proteins from CNF2-treated cells migrated slightly more slowly in SDS/PAGE than did the labeled proteins from the control cells. This shift in mobility of Rho proteins in SDS/PAGE was also observed when CNF2 and the RhoA protein were coexpressed in E. coli. We propose that Rho proteins are the targets of CNF2 in mammalian cells.

1. Because antiadrenergic therapy of hypertension may precipitate borderline cardiac compensation into failure, a method for detecting early cardiac insufficiency appeared desirable. 2. Eight out of thirty primary hypertensive patients were considered to be in incipient failure on the basis of the magnitude of the changes in left ventricular mean rates of systolic ejection and isovolumic pressure development after acute digitalization. 3. Antiadrenergic therapy precipitated these patients into overt failure, but did not affect cardiac performance in the others. 4. Left ventricular response to digitalis seems a reliable test in estimating cardiac reserve and in predicting the likelihood of decompensation after antiadrenergic treatment of hypertension.

1. Propranolol, when used for treating arterial hypertension, may influence determinants of both cardiac and vascular function; the consequent changes in cardiac performance may result from the interaction of different and possibly opposite effects. 2. Cardiac function was investigated in fifty-four primary hypertensive men in the pretreatment state and after 3 weeks of propranolol therapy at a daily dose of 320 mg. 3. ß-Receptor blockade caused depression of pre-injection left ventricular function, which was unrelated to the direction and the extent of changes in peripheral circulation. 4. The ejection left ventricular function could be either depressed or improved depending on the direction to which treatment shifted the vascular resistance, and consequently, the impedance to left ventricular ejection. 5. Withdrawal of the adrenergic support is probably the major factor responsible for the poor ventricular adaptation to an augmented impedance.

The cytotoxic necrotizing factor type 1 (CNF1) fromE. coli causes necrosis in rabbit skin and multinucleation in cultured cells. Cells exposed to CNF1 were characterized by changes in actin organization, mainly consisting in the presence of well-developed stress fibers and membrane ruffles. The interaction of CNF1 with the cell cytoskeleton probably promotes the cell spreading and interferes with the cytokinesis, leading to the formation of giant multinucleated cells.

1. The elevated beta-receptor responsiveness to adrenergic stimuli makes subjects with the primary hyperkinetic cardiac syndrome ideal for studying the electrical and dynamic responses of the heart to sympathetic activation. 2. In twelve men presenting with the syndrome, the effects of mental arithmetic and painful (cold) stress on the cardiac inotropic state were tested and correlated with the concomitant electrocardiographic changes. 3. Arithmetic and cold evoked responses opposite and divergent from the base-line state: The former induced vasodilatation, enhancement of cardiac rate, output, contractility and deep T wave inversion; the latter caused vasoconstriction, cardiac depression and full restoration of repolarization. 4. The sympathetic outflow elicited by stress is not generalized, but selectively directed to different circulatory levels in relation to the stimulus at work; cardiac sympathetic stimulation or inhibition has opposite effects on the repolarization phase.

1. In 27 severe primary hypertensive patients nifedipine (10 mg), administered orally, induced prompt (–21% of control at 30 min) and persistent (–17% at 120 min) fall of mean arterial pressure mediated through reduction of peripheral vascular resistance with rise of cardiac output. 2. The sublingual route (nine cases) showed more rapid onset of action and equal antihypertensive effectiveness. 3. In five patients with hypertensive crisis and acute left ventricular failure, the drug strikingly reduced systemic and pulmonary arterial pressures and relieved pulmonary oedema. 4. Prompt efficacy, ease of administration, absence of important side effects indicate that nifedipine may be a useful therapeutic agent in severe hypertension and in critical conditions that require rapid lowering of blood pressure.

C. difficile toxin A causes the rounding up and the formation of surface blebs in normal rat intestinal crypt cells (IEC-6). These phenomena, together with the inhibition of DNA synthesis and the cell lysis, could be the mechanisms leading the toxin-treated cells to death.

Purpose: Cardiac deconditioning due to immobilization is a risk factor for cardiovascular disease. The physiology of cardiac adaptation to deconditioning has not been fully elucidated. The purpose of the present study was to assess the effects of 21-days of strict head-down (-6 degrees) bed-rest (BR) deconditioning on left ventricular (LV) dimensions and mass measured by MRI. Methods: Ten healthy men (mean age 32±6) were enrolled; the experiment was conducted at DLR (Koln, Germany) as part of the European Space Agency BR studies. Steady-state free precession MRI images (7mm thickness, no gap, no overlap) were obtained (Symphony 1.5T, Siemens) in a stack of short-axis views from LV base to LV apex, before (PRE), at the end of BR (HDT20), and four days after the BR conclusion (POST). Endocardial and epicardial semi-automated contouring was performed using freely available software (Segment). Results: At HDT20, significant reductions in LV mass (16%), end-diastolic (26%) and end-systolic (27%) volumes and stroke volume (27%) were observed, while ejection fraction did not change. These changes were accompanied by a measured decrease (14%) in plasma and blood volume (by gas-rebreathing technique), as well as by a significant reduction (14%) in VO2max aerobic power, measured using a graded cycle ergometer test protocol to volitional fatigue, at one day after the BR conclusion, while expiratory exchange ratio did not change. At POST, LV volumes were restored, while LV mass was still trending towards control values. Conclusions: Cardiac adaptation to deconditioning affected LV mass and dimensions, as a combined result of LV remodeling and fluids loss, accompanied by worsening in aerobic power. This should be taken into account in patients with cardiovascular diseases, when immobilized in bed, to proper adjust the therapy, or to define appropriate physical exercises when possible, in order to avoid further complications.   Cardiac MRI parameters PREHDT20POSTLV mass (g)121±6102±11*114±16End-diastolic volume (ml)119±2590±14*118±25End-systolic volume (ml)42±831±8*45±14Stroke volume (ml)76±2259±11*73±15Ejection fraction (%)64±665±762±7 *: p<.01 vs PRE (one-way Anova for paired data and Tukey test)

Coronary angiography has diagnostic limitation in identifying non-stenotic coronary lesion (NSCL) responsible for ischemia. Myocardial perfusion defects in patients (pts) with NSCL have often been unreasonably considered by invasive cardiologists as being "false positive". We evaluated a prognostic value of gated SPECT MPI in unselected group of the pts with NSCL over a 24 month period of follow-up. 170 pts (115 males, 67.6%; age 42-68 years; mean age 56.4±9.2 years) with NSCL (stenosis of 50% or less of LAD and 70% or less of any other coronary artery or its major branches) were enrolled into the study. Retrospective analysis of 86 pts with NSCL and subsequent positive MPS performed within 6 months from the time of coronary angiography (study group) and 84 pts with normal scan results (control group) was performed. Follow-up period was for 24 months from the time of MPI or up to the time of major coronary event (MCE) - first occurrence of cardiac death or myocardial infarction. Over a two-year follow-up, approximately 11% of the pts in study group had MCE as compared to 3.2% in the control group (P < .01). Abnormal MPI, EF < 35% and high levels of hs-CRP were independent predictors for MCE in the study group. In multivariate analysis only an abnormal MPI remained to be an independent predictor regardless of size or severity of perfusion abnormalities (P <.005). Pts with NSCL on coronary angiography and myocardial perfusion defects have relatively high event rate (11%) of MCE over a period of 24 months from the time of MPI. So, we highly recommend gated SPECT MPI to be obligatory performed in all cases of NSCL to avoid life-threatening coronary complications in forthcoming future.

Purpose: Transthoracic 3D echocardiography (3DE) allows an unparalleled opportunity for quantifying the dynamic changes of the tricuspid annulus (TA). Accordingly, our aims were: (I) to assess the determinants of TA size during cardiac cycle in healthy subjects; (II) to propose an approach and timing for TA sizing using 3DE. Methods: In 50 healthy volunteers (45±14 yrs, range 18-74, 27 males, with no risk factors, symptoms, signs or history of cardiovascular disease and on no medication), a full-volume dataset of the right ventricle (RV) containing the tricuspid valve (TV) was acquired (Vivid E9, GE Healthcare). TA diameters (septo-lateral, SL; antero-posterior, AP) and areas were measured on multiplanar images (Flexi-slice, EchoPac BT12, GE Healthcare) at 5 time points during the cardiac cycle: OS (onset of systole, at TV closure); MS (mid-systole); ES (end-systole); ED (onset of diastole); LD (late diastole, after the P wave). RV volumes and ejection fraction (EF) were analyzed with commercial software (4D RV analysis, TomTec, D). Results: Temporal resolution of the 3D datasets was 32±4 vps (range 24-53). TA areas were more closely correlated with RV volumes and body surface area (BSA) than with either SL or AP diameters. TA areas increased during systole from OS (3.9±0.6 cm2/m2) to ES (4.9±0.8 cm2/m2) and reached its largest area in LD (6.7±1.0 cm2/m2). All 5 TA areas were correlated with BSA (r range 0.57-0.62) and RV volumes (r ranges 0.53-0.60 for end-diastolic volume and 0.43-0.50 for end-systolic volume, p<0.0001 for all). Indexed TA areas were not related to either age or gender. With multivariable analysis, both RV end-diastolic volume and BSA determined TA areas during systole and early diastole, while TA area at LD and at OS were independently related with BSA only. Conclusions: In healthy subjects, the main determinants of TA size are RV volume and BSA. The largest TA area occurs at LD and is independently related with BSA only. Therefore, normative values should be based on TA areas measured at LD and indexed for BSA. However, the rapid change in TA areas occurring from LD to OS underscores the importance of adequate temporal resolution of 3DE data sets for reliable TA measurements.