A new low molecular weight, MnII-containing scavenger of superoxide anion protects cardiac muscle cells from hypoxia/reoxygenation injury.

Reperfusion injury after oxygen starvation is a key pathogenic step in ischemic diseases. It mainly consists in oxidative stress, related to mitochondrial derangement and enhanced generation of reactive oxygen species (ROS), mainly superoxide anion (O2(•2)), and peroxynitrite by cells exposed to hypoxia. This in vitro study evaluates whether Mn(II)(4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-diacetate).2H2O, or Mn(II)(Me2DO2A), a new low molecular weight, Mn(II)-containing O2(•) scavenger, has a direct protective action on H9c2 rat cardiac muscle cells subjected to hypoxia and reoxygenation. Mn(II)(Me2DO2A) (1 and 10 µmol/l) was added to the culture medium at reoxygenation and maintained for 2 h. In parallel experiments, the inactive congener Zn(II)(Me2DO2A), in which Zn(II) replaced the functional Mn(II) center in the same organic scaffold, was used as negative control. Mn(II)(Me2DO2A) (10 µmol/l) significantly increased cardiac muscle cell viability (trypan blue assay), improved mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test, membrane potential Δψ), reduced apoptosis (mitochondrial permeability transition pore opening, caspase-3, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay), decreased intracellular ROS levels (2',7'-dichlorodihydrofluorescein diacetate and MitoSOX assays), and decreased protein nitroxidation (nitrotyrosine [NT] expression) and DNA oxidation (8-hydroxy-deoxyguanosine levels). Of note, Zn(II)(Me2DO2A) had no protective effect. The mechanism of Mn(II)(Me2DO2A) relies on concentration-dependent removal of harmful O2(•) generated at reoxygenation from dysfunctional mitochondria in hypoxia-induced cells, as indicated by the MitoSOX assay. This study suggests that Mn(II)(Me2DO2A) is a promising antioxidant drug capable of reducing O2(•)-mediated cell oxidative stress which occurs at reoxygenation after hypoxia. In perspective, Mn(II)(Me2DO2A) might be used to reduce ischemia-reperfusion organ damage in acute vascular diseases, as well as to extend the viability of explanted organs before transplantation.

The use of a non cultured autologous cell suspension to repair chronic ulcers.

The purpose of this uncontrolled study was to evaluate the clinical effectiveness of ReCell(®) system in the treatment of chronic ulcers. From October 2011 to July 2012, 20 patients, 8 men and 12 women with a mean age of 70 years, with chronic ulcers of different aetiology that were unresponsive to conventional therapies were recruited and treated using the ReCell(®) system. Patient pain rate, scar aesthetics and patient satisfaction were assessed using a Visual Analogue Scale, Manchester Scar Scale and the Rosenberg Self-Esteem Scale, respectively. Complete ulcer healing, defined as 100% reepithelialisation was observed between 40 and 60 days in 14 patients (70%) depending on the type of ulcer and comorbidity. At day 60 postprocedure, 80% reepithelialisation was present in five patients (25%), while one patient with concomitant psoriasis had 50% reepithelialisation. Pain scores improved by day 7 postprocedure. The function and aesthetics of the ReCell-treated patients were good. It is concluded that the ReCell technique may have provided the regenerative tissue stimulation necessary for the rapid healing of chronic ulcers, including those not responsive to more traditional methods.

Effects of a selective histamine H4R antagonist on inflammation in a model of carrageenan-induced pleurisy in the rat.

The histamine H4 receptor (H4R), recently cloned and identified, is a G-protein coupled histamine receptor family expressed in immune cells which plays an important role in inflammation. Recent data evidentiated that H4R antagonists can decrease airway inflammation and hyperreactivity in animal models of asthma. In the present study we evaluated the effect of the selective H4R antagonist JNJ7777120 (JNJ) in carrageenan-induced pleurisy, an in vivo model of inflammation, well characterized for cellular and molecular mechanisms. Intra-pleural administration of λ-carrageenan (1% w/v in 0.2 ml sterile saline) determined an intense recruitment of leucocytes in pleural exudates and in lung tissues, activated inducible nitric oxide (NO) synthase and cyclooxygenase-2, thus increasing the generation of harmful autacoids such as NO and pro-inflammatory prostaglandins, PgE2 and 6-ketoPgF(1α), increased cellular and DNA oxidative stress, measured as malondialdehyde and 8-OH-deoxyguanosine and the local generation of IL-1ß and TNF-α. Moreover, the activity of caspase-3, an early marker of apoptosis was also activated by λ-carrageenan injection. The pre-treatment with JNJ (5-10 mg Kg⁻¹ b.wt., given intrapleurally), 60 min before carrageenan markedly reduced all the studied parameters. This study clearly demonstrated that histamine H4R antagonists have anti-inflammatory effects and could have potential therapeutic application for the treatment of inflammatory diseases.

Treatment of stable vitiligo hands by ReCell system: a preliminary report.

BACKGROUND: The aim of this report was to analyze the results obtained with the ReCell system for the surgical treatment of stable vitiligo hands. MATERIALS AND METHODS: One patient with stable vitiligo of the hands was admitted at the Department of Plastic and Reconstructive Surgery, University of Rome Tor Vergata. The patient underwent to ReCell system for the treatment of stable vitiligo hands. RESULTS: The repigmentation was assessed using the Vitiligo Area Scoring index (VASI). The extent of pigmentation was scored as excellent, good, fair, and poor depending on the percentage of the repigmentation in the previously depigmented site. The color of the repigmented area was compared with the adjacent normally pigmented area. The patient had an excellent repigmentation. CONCLUSIONS: ReCell system is a simple, safe and feasible technique. The method that uses noncultured autologous epidermal suspension is simpler, cheaper, less time consuming and does not require sophisticated laboratory facilities, when compared with methods employing cultured melanocytes.

A case of labial fusion and urinary pseudo-incontinence in an elderly woman. A surgical treatment and a review.

Labial fusion is defined as either partial or complete adherence of the labia minora (1), and also called vulvar fusion, adhesions of the labia minor or conglutination of the labia minora and sinechia of the vulva. The complete and severe labial fusion is a rare pathology with a small number of cases reported in the literature in adults. We present a case report of a postmenopausal woman who presented with voiding difficulty and incontinence and was treated by surgical division of the adhesions and immediate resolution of the urinary incontinence confirmed by multichannel urodynamic test postoperatively.

A single heterozygous nucleotide substitution displays two different altered mechanisms in the FBN1 gene of five Italian Marfan patients.

The Fibrillin-1 gene (FBN1; chromosome 15q21.1) encodes a major glycoprotein component of the extracellular matrix. Mutations in FBN1, TGFBR1, TGFBR2 are known to cause Marfan syndrome (MIM 154700), a pleiotropic disorder. In the present study, we describe five novel missense FBN1 mutations in five Marfan patients that have the peculiarity to activate two contemporary mutational mechanisms: a missense mutation and exon skipping.

Use of platelet rich plasma and hyaluronic acid on exposed tendons of the foot and ankle.

Platelet rich plasma was used as an autologous scaffold for cellular growth, in combination with hyaluronic acid as a temporary dermal substitute. This aided healing of acute and chronic open wounds of the foot and ankle.

Use of a novel autologous cell-harvesting device to promote epithelialization and enhance appropriate pigmentation in scar reconstruction.

BACKGROUND: Epidermal replacement is an important step in the management of patients with post-traumatic and iatrogenic scars. Skin-colour variation from disease or trauma causes significant changes in self-image and appearance. AIM: The aim of our study was to analyse the results obtained with a novel autologous cell-harvesting system (ReCell) for epidermal replacement in patients with post-traumatic scars that had not improved with any other surgical procedure. METHODS: We recruited 30 patients with post-traumatic or iatrogenic scars admitted to our department over 2 years. The primary endpoints of the study were: (i) time for complete epithelialization (both treated area and biopsy site) and (ii) aesthetic and functional quality of the epitheliaization (colour, joint contractures). Infections, inflammations or any adverse effects of the procedure were also reported. RESULTS: In total, 30 patients were analysed. The aesthetic and functional outcomes were rated by both patient and surgeon. Pigmentation was rated by the Vancouver Scar Scale. Most (80%) of the patients had an excellent or good outcome, with pigmentation rated as normal in 60% of the group. CONCLUSIONS: The procedure is a feasible, simple and safe technique. It gives similar results to skin grafting but because it harvests from much smaller areas, can open possible future applications in the management of patients with large scars.

PPARgamma promoter polymorphisms and acute coronary syndrome.

BACKGROUND: PPARgamma (PPARg) is a nuclear transcription factor involved in the control of lipid and glucose homeostasis. Two PPARg common polymorphisms, Pro12Ala and 161C>T, have been found to be associated with cardiovascular disease. In this study, in addition to PPARg coding region, we looked for genetic variations in promoters and their association with acute coronary syndrome (ACS). METHODS: We studied 202 Italian patients with ACS, and 295 healthy Italian subjects by dHPLC (denaturing high-performance liquid chromatography), heteroduplex analysis and direct sequencing or RFLP (restriction fragment length polymorphism) analysis for screening mutations. RESULTS: We identified 7 new and 2 already published polymorphisms in PPARg promoters. The C>T93695 (promoter 4) mutation showed significantly different genotype distribution and allele frequency between controls and ACS patients (p<0.001); the T allele conferred a protection against ACS at both univariate (OR: 0.45, 95% CI 0.29-0.69: p<0.001) and multivariate analysis adjusted for sex, age and traditional cardiovascular risk factors (OR: 0.44, 95% CI 0.25-0.76: p<0.005). Moreover, the 161C>T polymorphism allele frequency (p=0.03) and genotype distribution (p=0.015) resulted to be different in ACS group if compared to healthy controls. CONCLUSIONS: The protective role of 93695C>T polymorphism in PPARg promoter in ACS suggests that PPARg genetic variants may affect the susceptibility to atherosclerotic diseases.

FBN1 mutation screening of patients with Marfan syndrome and related disorders: detection of 46 novel FBN1 mutations.

Fibrillin-1 gene (FBN1) mutations cause Marfan syndrome (MFS), an inherited connective tissue disorder with autosomal dominant transmission. Major clinical manifestations affect cardiovascular and skeletal apparatuses and ocular and central nervous systems. We analyzed FBN1 gene in 99 patients referred to our Center for Marfan Syndrome and Related Disorders (University of Florence, Florence, Italy): 85 were affected by MFS and 14 by other fibrillinopathies type I. We identified mutations in 80 patients. Among the 77 different mutational events, 46 had not been previously reported. They are represented by 49 missense (61%), 1 silent (1%), 13 nonsense (16%), 6 donor splice site mutations (8%), 8 small deletions (10%), and 3 small duplications (4%). The majority of missense mutations were within the calcium-binding epidermal growth factor-like domains. We found preferential associations between The Cys-missense mutations and ectopia lentis and premature termination codon mutations and skeletal manifestations. In contrast to what reported in literature, the cardiovascular system is severely affected also in patients carrying mutations in exons 1-10 and 59-65. In conclusion, we were able to detect FBN1 mutations in 88% of patients with MFS and in 36% of patients with other fibrillinopathies type I, confirming that FBN1 mutations are good predictors of classic MFS.

Detection of common and private mutations in the COL6A1 gene of patients with Bethlem myopathy.

BACKGROUND: Dominant mutations in COL6A1, COL6A2, and COL6A3, the three genes encoding collagen type VI, a ubiquitous extracellular matrix protein, are associated with Bethlem myopathy (BM) and Ullrich scleroatonic muscular dystrophy. METHODS: The authors devised a method to screen the entire coding sequence of the three genes by reverse transcriptase-PCR amplification of total RNA from skin fibroblasts and direct sequencing of the resulting 25 overlapping cDNA fragments covering 107 exons. RESULTS: Four splicing and four missense mutations were identified in 16 patients with BM, six of which are novel mutations in COL6A1. Both common and private mutations are localized in the alpha1 (VI) chain between the regions corresponding to the 3' end of the NH2-globular domain and the 5' end of the triple helix, encoded by exons 3 through 14. CONCLUSIONS: The clustering of the mutations in a relatively narrow area of the three collagen type VI chains in patients with Bethlem myopathy (BM) suggests that mutations in different regions could result in different phenotypes or in no phenotype at all. Moreover, the detection of mutations in only 60% of the patients suggests the existence of at least another gene associated with BM. The authors propose the direct sequencing of COL6 cDNAs as the first mutation screening analysis in BM, given the high number of exon-skipping events.

Cytokine gene expression and production by human LPS-stimulated mononuclear cells are inhibited by sulfated heparin-like semi-synthetic derivatives.

BACKGROUND: The K5 polysaccharide obtained from Escherichia coli strain 010:K5:H4 is a polymer of the disaccharidic unit formed by D-glucuronic acid and N-acetylglucosamine. This structure is akin to N-acetylheparosan, the precursory polymer of heparin and of heparan sulfate. This structural affinity with N-acetylated heparin and with de-sulfated heparin makes the K5 polysaccharide extremely useful for the preparation of sulfated heparin-like semi-synthetic derivatives. It has been demonstrated that heparins are able to inhibit tissue factor and cytokine production and expression by human monocytes. OBJECTIVE: The aim of this study was to evaluate the effects of four different heparin-like semi-synthetic derivatives on inflammatory cytokine production and expression by human mononuclear cells. RESULTS: The simultaneous addition of lipopolysaccharide (LPS; 0.2 and 10 micro g mL(-1)) and the K5 polysaccharide did not inhibit interleukin (IL)-1beta, IL-6 or tumor necrosis factor (TNF)-alpha production by stimulated mononuclear cells. IL-1beta, IL-6 and TNF-alpha concentrations in supernatants of LPS-stimulated mononuclear cells were not influenced by the addition of N,O-sulfated K5 polysaccharide (K5-N, OS) and epimerized N-sulfated K5 polysaccharide (K5 NS epi) at 5 and 10 microg mL(-1), whereas the addition of epimerized N,O-sulfated K5 polysaccharide (K5-N, OS epi) (5 and 10 microg mL(-1)) and O-sulfated K5 polysaccharide (K5-OS) (5 and 10 microg mL(-1)) to LPS-stimulated cells caused a significant dose-dependent inhibition of IL-1beta, IL-6 and TNF-alpha. All sulfated heparin-like semi-synthetic derivatives did not influence the IL-10 production by LPS-stimulated mononuclear cells. In LPS-stimulated cells (0.2 and 10 microg mL(-1)), K5-OS or K5-N, OS epi at 5 and 10 microg mL(-1) markedly decreased TNF-alpha mRNA expression. CONCLUSIONS: These results indicate that the sulfated heparin-like semi-synthetic derivatives K5-OS and K5-N, OS epi are able to inhibit both expression and production of inflammatory cytokines, whereas they do not influence the anti-inflammatory cytokine IL-10, suggesting a potential role for these products as modulators of inflammatory reactions.

New technique for optical control of the first trocar insertion.

BACKGROUND: In laparoscopic surgery serious complications caused by the blind insertion of the first trocar have been reported even after the pneumoperitoneum is established by means of a Veress needle. As a consequence, some techniques to safely insert the first port have been developed. Many surgeons advocate a minilaparotomy to position the first port. However, especially in obese patients, the dissection to insert the Hasson's trocar may be difficult and time consuming. As an alternative, optical disposable trocars have been studied and produced to be safely positioned under direct visualization. METHODS: A new technique to insert, under adequate optical control, a non disposable standard port is described. Personal experience in 126 consecutive patients is reviewed. Data were recorded prospectically and insertion time was recorded in every case. RESULTS: Medium time of trocar insertion was 55 sec (range 30-160 sec). Identification of distinct layers of the abdominal wall was always possible. There were no complications related to the insertion of the first reusable cannula under visual control. CONCLUSIONS: This technique may be considered a safe, simple and cost effective method to insert the first trocar.

Laparoscopic-assisted Hartmann's reversal with the Dexterity Pneumo Sleeve.

Several laparoscopic techniques of Hartmann reversal have been published. An alternative laparoscopic-assisted procedure, essentially aseptic, which uses the minilaparotomy for colostomy mobilization to apply the Dexterity Pneumo Sleeve device is described. This technique combines the advantages of the minimally-invasive approach with the direct access of the surgeon's hand to the patient's abdomen.

[Problems inherent in the diagnosis and treatment of common bile duct calculi during video-laparoscopic cholecystectomy. Report on 700 cases, February 1991 to December 1997]. / Problematiche inerenti alla diagnosi e al trattamento della calcolosi della via biliare principale in corso di videolaparocolecistectomia. Revisione di un'esperienza di 700 casi dal febbraio 1991 al dicembre 1997.

Conditions of predictivity seem to make treatment of common bile duct (CBD) stones during VLC more safe and adequate. IV cholangiography (or, nowadays, MRI and echoendoscopy) drive to a selective use of ERCP and cholangiography, help to reduce the incidence of residual lithiasis and give a "topographical map" of the CBD that can be useful, during VLC, in the search of the hilum structures, especially when the anatomical situation is not clear.

[Limitations of laparoscopic cholecystectomy]. / Limiti della colecistectomia laparoscopica.

Laparoscopic cholecystectomy has brought about an important and to some extent provocative revolution introducing the new dimension of minimally invasive surgery. The intrinsic limits of this new technique are analysed, as well as those that need to be established for its adoption to respect the rules of traditional surgery. Author's experience suggests that the explorative stage is widely indicated as a first step. This does not preclude early, reasoned and cautious conversion to laparotomy, but allows a safe and more generous use of the technique thanks to the possibility to check on patient selection.