Pneumonectomy: quality of life and long-term results.

AIM: Pneumonectomy is the standard surgery for resectable locally advanced lung cancer. Objectives of this study were: 1) to assess the overall survival; 2) to evaluate the pulmonary and cardiac function impairment; 3) to monitor quality of life (QoL) in a consecutive series of patients undergoing pneumonectomy, defining the potential risk factors of a poor prognosis. METHODS: From January 2003 to March 2010, 71 patients undergoing pneumonectomy for lung cancer or mesothelioma were prospectively enrolled in this study. Twenty-six patients underwent right pneumonectomy (2 of them underwent intrapericardial pneumonectomy), 31 left pneumonectomy (3 of them underwent intrapericardial pneumonectomy), 3 extended pneumonectomy, 3 extrapleural pneumonectomy and 5 patients underwent completion pneumonectomy. Three patients were not included in the study for early postoperative deaths (4.3%). All patients underwent complete preoperative assessment and one year after surgery. QoL was assessed by a questionnaire. RESULTS: One and five-year survival rate was 93% (N.=63) and 20% (N.=14), respectively. Mean values of FEV1 decreased from 2.59±0.75 L to 1.8±0.72 L (P<0.001). One year after surgery all patients showed moderate tricuspid valve insufficiency, PASP significantly higher and right ventricular free wall thickness moderately increased. An increased negative effect was recorded in the QoL scores with P<0.001. Three clinical and surgical parameters were identified as risk or protective factors for the survival outcome. CONCLUSION: Postoperative mortality (4.3%) and five-year survival (20%) after pneumonectomy seem to be satisfactory. Late cardiopulmonary insufficiency is uncommon and acceptable QoL is still achievable.

Human AB serum for generation of mesenchymal stem cells from human chorionic villi: comparison with other source and other media including platelet lysate.

OBJECTIVES: We have investigated foetal mesenchymal stem cells (MSCs) obtained from first-trimester chorionic villi (CV) and second-trimester amniotic fluid (AF), comparing them to adult bone marrow-derived MSCs. MATERIALS AND METHODS: We report on cell population growth in human allogeneic serum (HS) and platelet lysate (PL), immunophenotype, cytokine expression profile and immunoregulatory activity, of these foetal MSCs on stimulated peripheral blood mononuclear and lymphocyte subpopulations. RESULTS: Chorionic villi cells grow rapidly in HS, with 20 populations doublings (PDs) after 59 days (six passages), and also in animal serum, with 27 PDs after 65 days (seven passages). PL allowed for expansion in 60% of the samples tested, although it was lower than in HS. HS supported an average of 40 PDs of expansion in 20% of AF cells after 90 days, whereas animal serum supported 28.5 PDs in 66 days. CV and AF cells inhibited proliferation of stimulated T lymphocytes, suppressing population growth of both CD4+ and CD8+ T subpopulations and sometimes also, CD19+ cells. CONCLUSIONS: Our results indicate that CV would be an optimal source of MSCs with high expansion potential in a HS propagation system and immunoregulatory capacity of T and B lymphocytes. More than 90% of CV samples achieved large-scale expansion in HS, which is encouraging for potential clinical applications of these cells.

[T4 lung cancer: results of surgical treatment]. / Il tumore polmonare T4: risultati del trattamento chirurgico.

Stage T4 non small cell lung cancer (NSCLC) includes an heterogeneous group of locally advanced tumors. Results of surgery alone and of chemo and/or radiotherapy are disappointing with 5-year survival rates under 10%. Although palliative chemo-radiotherapy is the treatment of choice in most cases, radical resection has shown prognostic benefit in selected groups of patients with tumor infiltrating Superior Vena Cava, carina, aorta, left atrium and vertebral bodies. Completeness of resection and absence of mediastinal nodal involvement are fundamental conditions for the long-term success of surgery. Increased postoperative 30-day mortality and 90-day mortality rates have been reported up to 8% and 18% respectively. Neoadjuvant therapy, in the last decades, has shown to improve survival of T4 NSCLC patients undergoing surgery and to increase the number of patients suitable for surgical resection. Surgical resection is not indicated in patients with neoplastic pleural effusion since it is generally related to a worse prognosis in such cases. Conversely, patients with T4 tumor due to neoplastic satellite nodule in the same lobe are good surgical candidates. In some studies, these patients show a significant survival advantage after surgical treatment with respect to patients with other types of T4 tumors, when no mediastinal nodal involvement is associated.

Autologus platelet gel for the management of persistent alveolar fistula after lung resection.

Postoperative alveolar fistula (AF) associated with pleural cavity (PC) is a serious complication and a therapeutic challenge in thoracic surgery. The purpose of this study was to assess the efficacy of the use of the autologous platelet gel for the treatment of AF and PC. We treated a patient with post lung resection persistent alveolar fistula using a autologous platelet gel, a cellular compose produces at the Division of Immunohaematoligy and Trasfusion. The platelet gel-PRP (Platelet-Rich Plasma) is a biological material made of autologous platelets, extracted from a small amount of the patient's blood, centrifuged at 1100 g for 9 min. The PRP obtained was activated by addition of autologous thrombin and calcium chloride to form a matrix of fibrin (PRFM) thick. The patient presented important air leak after middle lobe wedge resection for solitary lung lesion with standard open decortication for important pleural adhesions post pleuritis. On postoperative day XIII the patient developed a thoracic empyema and consequently underwent a antibiotic pleural irrigation through the chest drainage based on the microbiological analysis of the pleural fluid. After a week we obtained the resolution of the empyema but a residual space remained and air leak persisted. We treated the patient with autologous platelet gel. We administer 7.5 mL of the autologous platelet gel across the chest drainage ever 72 hours for 3 times. After the third application we had the closure of the cavity and the cessation of air leak. Autologous platelet gel is easy to use, safe and inexpensive. It can be considered a valid therapeutic option in selected patients with a alveolar fistula and a lung partial re-expansion. The product consist of a significant amount of cellular components with healing anti-inflammatory an proregenerative properities that permit the body to heal tissue wounds faster and more efficiently. A sterile pleural cavity is fundamental conditions for the final success of the procedure.

Selection of CD271(+) cells and human AB serum allows a large expansion of mesenchymal stromal cells from human bone marrow.

BACKGROUND: Mesenchymal stromal cells (MSC) are promising candidates for cell therapy and tissue engineering and may be used to treat acute graft-versus-host disease (GvHD). However, major obstacles for their clinical use are the required cell dose and the biosafety and potential immunogenicity of fetal bovine serum (FBS), which is a crucial supplement of all media currently used for the culture of MSC. METHODS: In this study MSC were successfully expanded after selection of CD271 cells from human bone marrow (BM) mononuclear cells in medium supplemented with 10% pooled allogeneic human serum. RESULTS: We isolated MSC from 10 healthy donor BM by plastic adherence and immunomagnetic selection of the CD271(+) fraction and expanded MSC in medium supplemented with pooled human allogeneic serum and animal serum. We isolated a homogeneous multipotent population by CD271(+) selection with a proliferation rate that was higher than MSC isolated by plastic adherence, 6.8+/-1.57 compared with 2.07+/-1.40 logs. Similar to cells generated in animal serum medium, MSC from allogeneic human serum were positive for mesenchymal markers and negative for hematopoietic markers; moreover they expressed embryonic stem cell genes. A normal karyotype and differentiation capacity into adipogenic, osteogenic and chondrogenic lineages and neurosphere-like structures were preserved throughout long-term culture. DISCUSSION: Expansion of MSC is both feasible and large with a CD271-selected population in medium supplemented with 10% pooled allogeneic human serum, without loss of multipotent differentiation capacity or karyotype alterations.

Characterization and expansion of mesenchymal progenitor cells from first-trimester chorionic villi of human placenta.

BACKGROUND: Mesenchymal stromal cells (MSC) have been identified in a variety of fetal and adult tissues, including bone marrow (BM), fetal blood and liver. We report on the isolation, expansion and differentiation in vitro of MSC-like cells from chorionic villi (CV). METHODS: We evaluated 10 samples of CV collected at the first trimester (gestational age 11-13 weeks). We only used cells taken from back-up culture after a successful karyotype analysis. CV cells were characterized by morphologic, immunophenotypic and molecular analysis. The differentiation ability of mesenchymal and neural lineages was detected using specific culture conditions. Cell expansion was assessed after plating cells at different densities in different media, supplemented with animal and human serum. RESULTS: CV cells showed a homogeneous population of spindle-shaped cells after the first passage. Cells expressed CD90, CD105, CD73, CD44, CD29 and CD13 but not CD45, CD14, CD34 and CD117. They expressed Oct-4, Rex-1, GATA-4 and nestin, which characterize the undifferentiated stem cell state. They differentiated into osteocytes, adipocytes, chondrocytes and neuronal cells. Cell expansion was greater than that of adult BM-derived MSC, 9 logs with fetal bovine serum and 6 logs with human serum. Despite their high proliferative capacity, we did not observe any karyotypic abnormalities after culture. DISCUSSION: Our study shows that CV cells have better potential for expansion than adult stem cells. They can proliferate in a medium with human allogeneic serum and can differentiate into mesenchymal and neural lineages. CV cells may be an excellent cell source for therapeutic applications.

Neutrophil apoptosis in autoimmune Fas-defective MRL lpr/lpr mice.

The apoptosis-defective lpr (fas) mutation in MRL mice causes the early onset of a lupus-like autoimmune disease with concomitant inflammation. In order to analyse the consequences of the impaired Fas-dependent apoptosis on inflammation, the susceptibility to apoptosis of polymorphonuclear leukocytes (PMN), obtained from MRL lpr/lpr mice, has been studied. Peritoneal PMN from lpr/lpr and control (+/+) mice were recruited with a mild inflammatory stimulus. The number of cells collected from the peritoneal cavity of young lpr/lpr mice was comparable to that obtained from age-matched control mice, indicating that PMN homeostasis is maintained regardless of the loss-of-function Fas mutation. Recruited neutrophils were exposed in culture to apoptosis-inducing stimuli. Treatment with agonist anti-Fas antibody increased apoptosis of +/+ PMN, but did not affect lpr/lpr PMN which do not express Fas on their surface. However, lpr/lpr PMN could undergo both spontaneous and stimulus-induced apoptosis in a fashion comparable to or higher than that of control +/+ mice. Analysis of mRNA expression revealed that lpr/lpr PMN have reduced expression of IL-18, whereas IL-1beta, IFNgamma, caspase 1 and caspase 3 are expressed at levels comparable to those of +/+ cells. However, caspase-3-like activity was higher in PMN from lpr/lpr mice than in +/+ cells, and correlated with enhanced apoptosis. It could be concluded that in young, uncompromised lpr/lpr mice, PMN homeostasis is still fully regulated through the involvement of Fas-independent, compensatory, apoptotic mechanisms. This could include an increased participation of caspase 3 in the apoptotic pathway, consequent to enhanced activation of the enzyme and to the decreased production of IL-18, which acts as a competitive caspase 3 substrate.

Balance between autocrine interleukin-1beta and caspases defines life versus death of polymorphonuclear cells.

The role of endogenous IL-1beta in regulating spontaneous and Fas-triggered apoptosis of human PMN has been studied in relation to the activity of the IL-1beta-generating enzyme ICE (caspase-1), an enzyme also involved in the mechanism of cell death. Upon in vitro culture, PMN undergo spontaneous apoptosis and express increasing levels of IL-1beta, caspase-1- and caspase-3-like enzymes. Endogenous IL-1beta protects PMN from apoptosis, since inhibition of either IL-1beta or caspase-1 activity can accelerate PMN apoptotic death. Thus, in spontaneous PMN apoptosis caspase-1 essentially plays an anti-apoptotic role by inducing maturation of protective IL-1beta, whereas other molecules are responsible of driving apoptosis. Upon Fas triggering, PMN apoptosis is greatly accelerated, in correlation with increased caspase activity, whereas IL-1beta production is not augmented. Inhibition of IL-1beta activity can increase Fas-induced apoptosis, whereas caspase-1 inhibitors are without significant effect. It is hypothesized that in Fas-induced PMN apoptosis caspase-1 has a double role: it can protect from apoptosis through generation of protective IL-1beta, as in spontaneous apoptosis, and it can also exert pro-apoptotic activity which counterbalances the protective effect and allows accelerated apoptosis.

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Inhibitory activity of IL-1 receptor antagonist depends on the balance between binding capacity for IL-1 receptor type 1 and IL-1 receptor type II.

A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.

Purification of human recombinant interleukin 1 receptor antagonist proteins upon Bacillus subtilis sporulation.

Human interleukin 1 receptor antagonist (IL-1ra) and IL-1ra mutants were constitutively expressed in recombinant Bacillus subtilis in endocellular and active form. In order to optimize the purification of the recombinant proteins, a new method has been developed. After bacterial growth in fermenter, release of recombinant protein was achieved by starvation-induced sporulation. The sporulation supernatant was recovered by centrifugation, filtered, and subjected sequentially to cation- and anion-exchange chromatography. Alternatively, the fermenter's contents were directly subjected to expanded bed adsorption on a Streamline cation-exchange column, thus avoiding the centrifugation and filtration steps. Up to 88 mg of biological active purified recombinant protein per liter of culture was obtained, with a 72-79% recovery and 98% purity, depending on the molecule. By using the method described here, it is possible to achieve a spontaneous release of recombinant proteins expressed endocellularly at high levels in B. subtilis without need of a cell breakage step. Thus, this method could allow purification of the endocellular recombinant protein as if it were secreted. Furthermore, when using the expanded bed adsorption, highly purified protein was obtained in only two steps after sporulation. Among the advantages of the method, one of the most relevant is the possibility of keeping the system closed up to completion of the first purification step.

Interaction between interleukin-1 and ciliary neurotrophic factor in the regulation of neuroblastoma cell functions.

Human neuroblastoma cells SK-N-SH express significant numbers of IL-1R type I on their surface, as detected by saturation binding and RT-PCR, and are responsive to IL-1beta activation by producing inflammatory cytokines IL-6 and IL-8. IL-1beta can also have an indirect effect on nervous cell functions, since it is able to modulate the stimulus-induced increase of intracellular Ca++ levels, one of the first steps of the cell activation mechanism. In fact, on SK-N-SH neuroblastoma cells, IL-1beta can inhibit the Ca++ increase induced by stimulation of acetylcholine receptors with carbachol. In parallel to IL-1beta, the neurotrophic factor CNTF also shows an inhibitory effect on carbachol-stimulated Ca++ increase in CNTFRalpha-expressing SK-N-SH cells. However, when simultaneously present, the two cytokines cross-inhibit, thus allowing full cell activation in response to the cholinoceptor agonist. The inhibitory effect of CNTF on IL-1beta activities on nervous cells was confirmed in the IL-6 production assay. In fact, while CNTF could not induce IL-6 production, it could strongly inhibit cytokine production in response to IL-1beta in SK-N-SH cells. The down-modulation of IL-1 effects by CNTF could be one of the mechanisms controlling the extent of the inflammatory reaction at the nervous system level.

Transfected type II interleukin-1 receptor impairs responsiveness of human keratinocytes to interleukin-1.

Of the two known types of specific receptors for interleukin (IL)-1, the function of the type II IL-1 receptor (IL-1RII) is still elusive. IL-1RII is allegedly devoid of signaling capacity and is therefore thought to act by trapping and inhibiting IL-1. To directly assess the functional role of IL-1RII, a human keratinocyte cell line has been stably transfected with a cDNA coding for IL-1RII, and its responsiveness to IL-1 has been compared with that of nontransfected cells. Parental cells express IL-1RI and are responsive to low doses of IL-1, whereas transfected cells overexpress IL-1RII, both in its membrane and soluble form, and show a dramatically impaired response to IL-1. Selective block of IL-1RII restores the ability of transfected keratinocytes to respond to IL-1, indicating that the overexpressed IL-1RII is in fact uniquely responsible for their refractoriness to IL-1. The main mechanism of unresponsiveness in transfected keratinocytes appears to be the capture and neutralization of IL-1 by the soluble form of IL-1RII.

Sporulation: an alternative way to recover recombinant proteins from Bacillus subtilis.

A recombinant strain of Bacillus subtilis engineered for endocellular expression of human interleukin-1 receptor antagonist (IL-Ira) was subjected to sporulation. The recombinant protein was recovered from the sporulation supernatant in quantities, purity, and activity comparable with those obtained from a traditional cell lysate. Thus, exploitation of this natural mechanism of autolysis could overcome problems of intact protein recovery related to the cell disruption step.

Conformational changes in pantetheine hydrolase as a function of guanidinium chloride concentration.

The denaturation of pantetheinase (pantetheine hydrolase, EC 3.5.1.-) was followed in guanidinium chloride using tyrosyl and tryptophanyl residues as probes in connection with change in enzymatic activity. Movements of tryptophanyl and tyrosyl residues during denaturation were studied by second-derivative and fluorescence spectroscopy and the number of these amino acids present in the protein was calculated from spectroscopic data. Pantetheinase shows a very high resistance to denaturation, being completely unfolded at guanidinium chloride concentration higher than 6.5 M. Monitoring enzymatic activity shows that inactivation of the enzyme occurred before noticeable conformational changes were detected and it is suggested that the conformation of the active site is flexible and easily perturbable compared to the protein as a whole. This inactivation is reversible, as shown by renaturation experiments. Second-derivative and fluorescence spectra showed also that tyrosyl and tryptophanyl residues are largely exposed in the native protein, confirming its hydrophobic behavior.

A kinetic study on pantetheinase inhibition by disulfides.

The mammalian enzyme pantetheinase, which hydrolyzes pantetheine to pantothenic acid and cysteamine, is inhibited by many thiol reagents and activated by thiols. Two thiol groups of different reactivity and accessibility are involved in the catalytic process [Ricci, G., Nardini, M., Chiaraluce, R., Duprè, S. & Cavallini, D. (1986) Biochim. Biophys. Acta 870, 82-91]. The inhibition kinetics by some natural and synthetic disulfides [pantethine, cystamine, 5,5'-dithiobis(2-nitrobenzoic acid), 4,4'-dithiodipyridine and oxidized mercaptoethanol] has been studied by two experimental approaches, either by monitoring activity after incubation of the enzyme with the inhibitor or by determining the progress curves in the presence of substrate and inhibitor. Data reported here indicate that pantetheinase reacts irreversibly with various disulfides in a time-dependent manner with the formation of a mixed disulfide apparently preceeded by a conformational change, giving a modified E* form with new kinetic parameters. This modified form may be further competitively inhibited by disulfides interacting with the enzyme at the active site.

Derivative spectroscopy as a nonperturbative tool to detect the state of liposome-entrapped sulbactam.

Sulbactam, a beta-lactam antibiotic, absorbs uv light at 273 nm when in alkaline media, whereas at neutral or acidic pH this peak disappears. Sulbactam-loaded liposomes, prepared by reverse-phase evaporation, were spectroscopically analyzed by the derivative mode measuring the peak-through amplitude between +258 and -285 nm, so that the spectral interference of the sample is eliminated. Taking advantage of this experimental approach, we could study the influence of different parameters on the dissociation state of the drug when entrapped in dipalmitoyl phosphatidylcholine liposomes. In particular, following the time course of sulbactam peak disappearance, we found that: (i) the effectiveness of protonating the sulbactam of the chosen buffers is acetate/acetic acid > succinate/succinic acid > citrate/citric acid; (ii) the rate of signal disappearance is influenced by the externally imposed pH and can be somewhat related to the dissociation state of the organic acids; (iii) as expected, the whole phenomenon is temperature dependent. The observations reported here might be the basis for quantitative permeation studies in synthetic and/or natural membranes using this methodology.

[Ileorectal anastomosis with a B.A.R. A clinical case report. Bowel anastomosis ring]. / Anastomosi ileo rettale con B.A.R. Descrizione di un caso clinico.

The device invented in 1892 by Murphy is now being used again: as a matter of fact, modern technologies allowed for the development of a pressure bowel-anastomosis system with complete reabsorption, whose mechanical structure resembles Murphy's device. In 1987, in digestive anastomosis, the B.A.R. (Bowel Anastomosis Ring) pressure suturing device was successfully used. In this case report, the authors, on the basis of previous experiences concerning pressure bowel anastomosis with biodegradable material, describe the use of the B.A.R. for the performance of an ileo-rectal sub-peritoneal anastomosis in a patient with rectal neoplasia and previous ileorectostomy. In the authors' opinion, the absence of post-surgery complications and the very good functional outcome that was achieved in such a complex case, from a pathological and surgical point of view, is a further contribution to the validity of such methodology, which is also supported by several studies carried out both in the U.S.A. and in Europe on colic and upper digestive surgery. By overcoming the last hindrance represented by the performance of esophageal anastomosis, it will be possible to consider B.A.R. as a useful and proper method to be generally applied in digestive surgery.