Cardiopulmonary testing in adult patients with ß-thalassemia major in comparison to healthy subjects.

ß-Thalassemia patients often have a reduced capacity of exercise and abnormal respiratory function parameters, but the reasons are unclear. In order to identify the causes of the exercise limitation, we performed a cardiopulmonary exercise testing (CPET) in a group of 54 adult ß-thalassemia major (TM) patients without pulmonary arterial hypertension and in a group of healthy control subjects. All subjects underwent cardiac echocardiography and carried out pulmonary function tests. TM patients also filled an IPAQ questionnaire on usual physical activity (PA).Overall, TM patients have a diminished exercise performance in comparison to control subjects. In fact, peak oxygen uptake (V'O2 peak), expressing maximum exercise capacity, was decreased in 81.5% of the patients; similarly, anaerobic threshold (V'O2@AT) and O2 pulse also resulted lowered. In multivariable regression models adjusted for gender, age, BMI, and mean haemoglobin, V'O2 peak and O2 pulse were positively associated with cardiac iron overload (T2*). No ventilatory limitation to exercise was observed. The most important causes of exercise limitation in these patients were muscular deconditioning and reduced cardiac inotropism due to iron deposition. Only 15/54 (27.8%) TM patients used to perform vigorous physical activity. These results suggest that a program of regular physical activity may be useful to increase the tolerance to effort and therefore to improve the quality of life in these patients.

Exacerbations and Pseudomonas aeruginosa colonization are associated with altered lung structure and function in primary ciliary dyskinesia.

BACKGROUND: Recurrent bacterial infections of the respiratory tract are one of the major clinical features of the primary ciliary dyskinesia (PCD), a rare genetic disease due to malfunctioning of motile cilia. Chronic infections and persistent inflammation of the respiratory system result in progressive lung disease. Aim of the study was to highlight the main factors associated with clinical, functional and anatomical deterioration in PCD patients. METHODS: We retrospectively analyzed data from 58 patients with PCD, 37 adults and 21 children. The demographic and clinical data, forced expiratory volume at 1 s (FEV1) and forced vital capacity (FVC), sputum microbiology and imaging results (chest CT scores-modified Bhalla) were recorded. Patients were stratified according to the number of exacerbations (< 2/year vs ≥ 2/year) and chronic Pseudomonas aeruginosa (PA) colonization. The possible correlations between lung function and chest CT scores were assessed; we also evaluated the correlation between these parameters and the severity scores for bronchiectasis (BSI, FACED and e-FACED). RESULTS: Chest CT scores showed a significant correlation with FEV1 (p = 0.0002), age (p <  0.0001), BMI (p = 0.0002) and number of lung lobes involved (p <  0.0001). PA colonization had an overall prevalence of 32.6%: no significant difference in FEV1 between PA colonized and non-colonized patients was found (p = 0.70), while chest CT score was significantly worse in chronic PA colonized patients (p = 0.009). Patients with a high number of exacerbation (≥ 2/year) were older (p = 0.01), had lower FEV1 (p = 0.03), greater number of lobes involved (p < 0.001) and worse CT score than patients with low number of exacerbations (p = 0.001); they also had higher prevalence of PA chronic bronchial infection (33.3% versus 13.6%, p = 0.10). Multivariable linear regression analyses adjusted for gender, age and BMI showed positive associations between PA colonization and number of exacerbations with severity of disease (number of lobes involved, CT score, BSI, FACED, and e-FACED). CONCLUSIONS: In our PCD population the number of exacerbations (≥ 2/year) and PA colonization were the two most relevant factors associated with severity of disease.

Emerging ciliopathies: are respiratory cilia compromised in Usher syndrome?

PURPOSE: Usher syndrome is a ciliopathy involving photoreceptors and cochlear hair cells (sensory cilia): since sensory and motor ciliopathies can overlap, we analysed the respiratory cilia (motile) in 17 patients affected by Usher syndrome and 18 healthy control subject. PATIENTS AND METHODS: We studied the mucociliary transport time with the saccharine test, ciliary motility and ultrastructure of respiratory cilia obtained by nasal brushing; we also recorded the classical respiratory function values by spirometry. RESULTS: All enrolled subjects showed normal respiratory function values. The mean mucociliary transport time with saccharine was 22.33 ± 17.96 min, which is in the range of normal values. The mean ciliary beat frequency of all subjects was 8.81 ± 2.18 Hz, which is a value approaching the lower physiological limit. None of the classical ciliary alterations characterizing the "ciliary primary dyskinesia" was detected, although two patients showed alterations in number and arrangement of peripheral microtubules and one patient had abnormal ciliary roots. CONCLUSIONS: Respiratory cilia in Usher patients don't seem to have evident ultrastructural alterations, as expected, but the fact that the ciliary motility appeared slightly reduced could emphasize that a rigid distinction between sensory and motor ciliopathies may not reflect what really occurs.

Colonization by Candida auris in critically ill patients: role of cutaneous and rectal localization during an outbreak.

BACKGROUND: Candida auris infections have been reported worldwide since the pathogen was isolated in 2009. AIM: To analyse the incidence of cutaneous and intestinal colonization, and connection with infections by the organism, in a hospital setting of a C. auris epidemic. METHODS: This was a retrospective study in intensive care units (ICUs) at a 1200-bed Italian hospital. The incidence of cutaneous positive swabs, and cutaneous carriers, for C. auris was compared to that of rectal positive swabs, and intestinal carriers, and both were correlated with C. auris infections. FINDINGS: A total of 399 patients were included. Seventy-seven patients were infected by C. auris. The ratio of C. auris positive skin swabs from screening in ICUs was 24%. The ratio obtained from infected patients and intestinal C. auris carriers was 49.1%, likewise rectal swabs from a similar cohort of patients (P = 0.373). Of this cohort, 39.7% and 5.5% were colonized only in skin and in rectum, respectively, while 54.8% was colonized in both sites. Of skin swabs, 12.3% and 83.6%, respectively, were always positive and variable over time in single subjects, while 31.5% and 41.1% of rectal swabs were always positive and variable (P = 0.000). Intestinal colonization was associated with increased risk for C. auris urinary infections (P = 0.006). CONCLUSION: C. auris intestinal carriers were fewer than cutaneous carriers, but more continuously colonized. Rectal and skin swabs can be good tools for surveillance, respectively, of colonization and of hygiene measures effectiveness. Urinary tract infections by C. auris appeared to increase along with gastrointestinal presence of the yeast.

Improved isolation of carbapenem-resistant Enterobacterales on selective-differential media extending the incubation time: an approach to strengthen the antimicrobial surveillance from rectal swabs.

BACKGROUND: Surveillance of carbapenem-resistant Enterobacterales (CRE) carriers is the first measure of hospital infection control. Screening of CRE carriage can be assessed through culture and molecular techniques, each with specific properties of turnaround-times, sensitivity and specificity. METHODS: This was a prospective study in a 1200-bed university hospital in Genoa, Italy, where CRE screening is performed analysing cultures from rectal swabs. Our 18-months intervention was to extend the incubation time of the corresponding plates from 48 to 288 h, after reporting negative tests, to evaluate the possible impact on the cultures. FINDINGS: A total of 362 patients giving 19,278 swabs and corresponding plates were included. After baseline incubation, plate positivity was 3%, while after the overall lengthened times it was 3.7%. Extended incubation was associated with change in the relative frequency of the most represented species. In particular, we observed reduced presence of total and resistant Klebsiella pneumoniae strains (P<0.001) and increased presence of Enterobacter cloacae complex, total and sensitive (P<0.001). By extending incubation time, a reduced frequency of overall Enterobacterales strains with high resistance to ertapenem (MIC ≥4 mg/L) was also found (P=0.005), particularly that of K. pneumoniae (P<0.001), while the presence of E. cloacae complex increased among organisms with low resistance levels to ertapenem (P<0.001). CONCLUSIONS: Extending the incubation time of the cultures increased the number of CREs grown, and expanded the bacterial scenario of rectal colonization through the recovery of poorly resistant strains and otherwise undetected species.

Selective delivery of pentamidine toward cancer cells by self-assembled nanoparticles.

Pentamidine (PTM) is an aromatic diamidine approved for the treatment of parasitic infections that has been recently proposed for possible repositioning as an anticancer drug. To this aim, efforts have been made to improve its therapeutic efficacy and reduce associated adverse effects through both covalent derivatization and association with nanocarriers. To efficiently encapsulate PTM into biocompatible nanoparticles and to enhance its selectivity toward cancer cells, a squalene (SQ) derivative (1,1',2-tris-norsqualenoic acid, SQ-COOH) was selected to prepare PTM-loaded nanocarriers. Indeed, SQ and its derivatives self-assemble into nanoparticles in aqueous media. Furthermore, SQ-bioconjugates strongly interact with low-density lipoproteins (LDL), thus favoring preferential accumulation in cells overexpressing the LDL receptor (LDLR). We report here the preparation of nanocarriers by ion-pairing between the negatively charged SQ-COOH and the positively charged PTM free base (PTM-B), which allowed the covalent grafting of SQ to PTM to be avoided. The nanoparticles were characterized (mean size < 200 nm and zeta potential < -20 mV for SQ-COOH/PTM-B 3:1 molar ratio) and molecular modelling studies of the SQ-COOH/PTM-B interaction confirmed the nanocarrier stability. Finally, the ability to indirectly target LDLR-overexpressing cancer cells was evaluated by in vitro cell viability assays and confirmed by LDLR silencing, serum privation and simvastatin treatment.

A novel Escherichia coli expression-export vector containing alkaline phosphatase as an insertional inactivation screening system.

An Escherichia coli expression-export vector was constructed (pCGV1, 6.3 kb) containing the alkaline phosphatase structural gene (phoA) located downstream from the phage lambda pR and pL promoters positioned in tandem and the cIts857 gene encoding lambda thermosensitive repressor. The phoA gene is fused to DNA encoding a hybrid signal sequence that contains the N-terminal portion of the beta-lactamase (Bla) signal sequence and the C-terminal region of the PhoA signal sequence. Within the DNA encoding hybrid signal sequence, a unique NheI restriction site is present where polymerase chain reaction (PCR)-amplified genes may be cloned. The 5' PCR primers reconstitute the C-terminal portion of either the PhoA or Bla signal sequences to restore an intact signal peptide. Recombinant phoA- clones are selected on indicator plates containing 5-bromo-4-chloro-3-indolyl phosphate.

Autologous unpurged bone marrow transplantation for acute non-lymphoblastic leukaemia in first complete remission.

Twenty-five patients with acute non-lymphoblastic leukemia (ANLL) in first complete remission underwent autologous bone marrow transplantation (ABMT) between March 1984 and March 1988. The high-dose therapy employed included cyclophosphamide followed by total body irradiation (10 Gy), administered as a single dose. The median time from complete remission to ABMT was 5 months (range 2-9 months). Thirteen (52%) patients remain in complete remission 10-51 months (median 25 months) after ABMT and 14-60 months (median 32 months) after achieving complete remission. Causes of death were recurrent leukemia (five patients), parenchymal toxicities (acute respiratory distress syndrome, veno-occlusive disease) (three patients), cerebral haemorrhage (one patient), cerebral aspergillosis (one patient) and viral hepatitis (one patient). Six patients relapsed at a median of 5 months after ABMT (range 4-10 months). In conclusion, this study has resulted in survival data comparable to those of other institutions and the best reported outcomes of conventional chemotherapy.

Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli.

Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot. The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (Ion protease- and heat shock protease-deficient). Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium. These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins.

The use of monoclonal antibodies for studying the biological properties of Staphylococcus aureus endo-beta-N-acetylglucosaminidase.

Staphylococcus aureus endo-beta-N-acetylglucosaminidase (SaG) has been suggested to function as a virulence determinant which interferes with the host cellular immune response. To further characterize the biological properties of SaG, monoclonal antibodies (mAbs) were raised against purified SaG. Four IgG1 subclass mAbs were obtained, none of which reacted with the reduced, sodium dodecyl sulphate pretreated or boiled enzyme. The ability of the mAbs to react with the enzymes present in supernatants obtained from 197 S. aureus strains indicated that they recognized epitopes which are highly conserved; bacteriolytic enzymes produced by staphylococci other than S. aureus did not show any cross-reactivity. After pretreatment of SaG with mAbs (mAb-SaG molar ratios varying from 1 to 20), it was shown that all selected mAbs caused, at a mAb:SaG molar ratio of 10, a 90% inhibition of SaG bacteriolytic activity and a statistically significant reduction of its ability to interfere with phagocytosis by human polymorphonuclear leukocytes. All selected mAbs reacted with several commercially available exo-beta-N-acetylglucosaminidases; mAb C1/10-11 also reacted with chicken and turkey egg muramidases and, at a mAb:SaG molar ratio of 10, inhibited their bacteriolytic activity by 97%. This suggests that one or more epitopes present in the above exo-glucosaminidases and muramidases share some degree of homology with others present in SaG.

A New Combination of Idarubicin, Etoposide and Cytarabine in Untreated Acute Non-Lymphoblastic Leukemia.

Sixty-seven unselected adult patients with untreated acute non lymphotblastic leukemia (ANLL) ranging in age from 15 to 80 years received a new induction regimen consisting of Idarubicin, Etoposide and Cytarabine. Patients who entered complete remission (CR) were then allocated to 4 courses of post remission intensification. After this, patients under 50 years of age with a compatible donor were given allogeneic bone marrow transplantation (BMT) or autologous BMT (ABMT) in those without an HLA-compatible donor; the remainder, older than 50, did not receive further therapy. Fifty-six of 67 patients (83.5%) achieved CR (02.5% in young and 70.3% in old patients) and 40 (71 %) after the first course. Seven patients (of whom, 6 were > 50 years) died in aplasia during the induction phase and four additional patients (all elderly) died during post-remission intensification without recurrent disease. Subsequently, the younger patients received transplants (BMT: 4 pts; ABMT: 10 pts). Twelve: of the 52 (23%) who survived post remission intensification (BMT: 1; ABMT: 4; others: 7) are disease free survivors 9-67 months (median, 32 months) after achieving CR. In conclusion, this intensive chemotherapy regimen is highly effective both in young and odder patients but the post-remission intensification may be too aggressive for elderly patients.

Identification of immunodominant epitopes in the filamentous hemagglutinin of Bordetella pertussis.

The filamentous hemagglutinin (FHA) of Bordetella pertussis is a principal adhesin, which plays a key role in the colonization of the upper respiratory tract. FHA is also a protective antigen, which has been incorporated in the new generation of acellular vaccines against whooping cough. The protein is synthesized as a large 367-kDa precursor, which is then processed into a 220-kDa secreted polypeptide. To optimize the use of this protein for vaccine purposes it would be helpful to define the regions encompassing immunodominant epitopes. Twelve recombinant plasmids have been generated encoding fusion proteins between fragments of the matured-secreted 220-kDa form of FHA and the vector-encoded phage MS2 polymerase. Protein extracts of the resulting recombinant clones have been tested for reactivity with sera from 20 patients convalescent from whooping cough, and two human standard sera. The results indicate the presence of an immunodominant B cell epitope in the polypeptide coded by a 1-kb DNA fragment encompassing positions 5781-6800 of the published sequence. These results suggest that the identified fragment should be conserved in the formulation of vaccines against pertussis.

Nasal ciliary function and ultrastructure in Down syndrome.

OBJECTIVES/HYPOTHESIS: To investigate the in vivo nasal ciliary beat and the ciliary ultrastructure in Down syndrome because, although in this condition an increased susceptibility to respiratory tract infections has been reported by several authors, the nature of this phenomenon is not fully understood. STUDY DESIGN: Experimental study of 18 subjects with Down syndrome and 18 healthy control subjects. METHODS: Ciliary beat frequency (CBF) was measured on samples of ciliated epithelium obtained from the inferior nasal turbinate; a further brushing for ultrastructural analysis was performed only in subjects showing a CBF reduction or a ciliary movement alteration. RESULTS: The mean CBF in the group with Down syndrome was 7 +/- 2.82 Hz, and in the control subjects it was 10.94 +/- 0.65 Hz. In the same 66.6% of subjects with Down syndrome, we observed a fibrillatory movement of cilia and no metachronicity was present. Moreover, in 14 subjects with Down syndrome as hyperproduction of mucus was present. Ultrastructural evaluation at transmission election microscopy instead revealed a normal architecture of cilia. CONCLUSION: We attribute the nature of the mucociliary defect in Down syndrome to recurrent respiratory tract infections causing changes in mucus properties as in rheological parameters and not to a primitive defect of cilia.

The effects of calcitonin nasal preparations and their excipients on mucociliary clearance in an ex-vivo frog palate test.

The topical tolerability of the commercial preparation of 1-7 Asu-eel and salmon calcitonin with 2% ammonium glycyrrhyzinate and 0.01% benzalkonium chloride, respectively, and of their excipients mixture in solution with increasing concentrations of ammonium glycyrrhyzinate and benzalkonium chloride, respectively, were assessed by investigating their effects on the mucociliary transport velocity in the ex-vivo frog palate preparation. This preparation provides an integrated biological model readily usable in the laboratory which closely resembles human nasal mucociliary clearance mechanism and can be used for rapid testing and toxicity of agents proposed for topical administration in the upper and lower airways. Frog-Ringer control, 1-7 Asu-eel and salmon calcitonin commercial spray preparations and the excipients plus 2% ammonium glycyrrhyzinate and plus 0.01% benzalkonium chloride did not modify significantly the mucociliary transport velocity, confirming their very good tolerability on ciliated epithelium. Higher concentrations of ammonium glycyrrhyzinate (10 and 20%) caused significant slowing, on average -32 and -55%, respectively. Higher concentrations of benzalkonium chloride (0.05 and 0.1%) also caused significant slowing, on average, -43.5 and -87%, respectively.

Sub-lethal concentrations of clarithromycin interfere with the expression of Staphylococcus aureus adhesiveness to human cells.

It has been known for some time that some antibiotics, generally at sub-lethal concentrations, are able to alter the morphology and the shape of bacteria. However, more subtle molecular alterations can also be present, such as disorganization of bacterial surface architecture, which leads to changes in the surface electrical charge that can influence the forces of attraction or repulsion responsible for interaction of bacterial surfaces with environmental surfaces. Bacterial adhesion to epithelial cells is a phenomenon regulated by these mechanisms. Clarithromycin, a new macrolide, at sub-inhibitory concentrations from 1/2 to 1/16 of the MIC, that is to say, from 0.12 to 0.015 microgram/ml, significantly reduces adhesion of Staphylococcus aureus to human buccal epithelial cells. Clarithromycin, as other antibiotics that interfere with the bacterial protein synthesis, should also be able to disturb the synthesis of adhesins. These are ligand molecules located on the surface of bacteria, and thus reduce the ability of bacteria to bind specifically to complementary molecules on the surfaces of epithelial cells which is necessary for host colonization.

The role of chemotherapy in polymorphonuclear phagocytosis in pneumonia.

The efficacy of the host defense system is a determining factor for the outcome of antimicrobial events in infected patients. The neutrophil granulocyte plays a key role in the lung's defense against bacterial invasion and in the absence of a sufficient attraction of functionally intact neutrophils in the lung, following bacterial challenge, severe pulmonary infection may result. The involvement of phagocytes in pneumonia is well known: infiltration of lung parenchyma by neutrophils occurs within a short time in response to infection and is followed by an influx of monocytes. We investigated the effects of antimicrobial therapy in pneumonia on polymorphonuclear neutrophil (PMN) phagocytosis.

Inhibition of bacterial adhesion by sub-inhibitory concentrations: brodimoprim vs trimethoprim.

There is accumulating evidence that sub-inhibitory concentrations (sub-MICs) of many antibiotics are not without effect on bacteria and even though they do not kill bacteria, they are still able to interfere with some important aspects of bacterial cell function. The aim of the present study was to investigate the effect of sub-MICs of brodimoprim and trimethoprim on Escherichia coli and Staphylococcus aureus adhesiveness to human mucosal cells. Sub-MICs of brodimoprim down to 1/32 MIC (0.03 microgram/ml) significantly reduced the E. coli adhesion to human buccal epithelial cells and this inhibition was significantly higher than the corresponding pattern for trimethoprim. Adhesion of S. aureus was significantly reduced down to 1/16 MIC for both brodimoprim and trimethoprim but no significant differences resulted between the two patterns. 2,4 Diaminopyrimidines and related structures have a high affinity for the enzyme dihydrofolate reductase and this causes a reduction in the synthesis of essential purines, thus reducing also DNA and the synthesis or expression of surface adhesins.

Thymomodulin stimulates phagocytosis in vitro by rat macrophages and human polymorphonuclear cells.

Activation of non-specific host defenses can increase resistance to infection in patients and especially those with reduced immune response. Thymomodulin is a calf thymic derivative containing low molecular weight peptides, which exerts immunomodulating activity probably through an enhancement of lymphocyte functions. To explore this possibility, rat macrophages (MP) and human polymorphonuclear (HPMN) cells were incubated in vitro with 100, 200, 400 micrograms/ml of thymomodulin at 37 degrees C for 60 min and their phagocytic activity was investigated. The number of phagocytosing cells was significantly increased following increasing concentrations of thymomodulin and the percentage of phagocytosis was increased more for human PMNs in comparison with rat MP, while the values of the phagocytic index were not modified after challenge with thymomodulin both for MPs and HPMNs.

Alterations in surface morphology of Candida albicans produced by rilopirox: a scanning electron microscopy study.

The topological changes produced in Candida albicans cells by incubation in vitro with rilopirox have been investigated by scanning electron microscopy. Rilopirox is a new hydroxypyridone compound with fungicidal activity and the effects of 1x MIC (2.9 micrograms/ml) and 4 x MIC (11.6 micrograms/ml) after 1, 12, 24 hours of incubation were evaluated. The morphological alterations produced by rilopirox are round shapes, collapsed cells, surface folds, clusters, holes and thorn-like extrusion. The effects of rilopirox are already evident at 1 x MIC and after 1 h but their frequency and severity are correlated with the time of incubation and the MIC.

Restoration of polymorphonuclear leukocyte function in elderly subjects by thymomodulin.

Senescence is a specific physiological evolution of human beings associated with a reduction in the functionality of several apparatuses, including the immune system. Thymomodulin (TMD) contains thymus polypeptides (< 10,000 D) and it has been used in a variety of disorders associated with defective immunological functions. The aim of this study was to investigate the effect on polymorphonuclear leukocyte (PMNs) phagocytosis and oxidative burst of a 6-week treatment with 160 mg/day TMD orally in elderly subjects (85.5 +/- 9.7 years). Elderly subjects have impaired PMN phagocytosis and the following release of oxidant radicals. Treatment with TMD for 6 weeks had a restoring effect; phagocytosis and the phagocytic index were significantly improved, with increases of 132.6% and 112.5%. These findings indicate that TMD might be given to enhance the immunodefenses of immunocompromised elderly subjects. Luminol-dependent chemiluminescence was increased by 15.6%, which was not significant, indicating a different response between phagocytosis and release of oxidant radicals.