Endothelin receptor antagonists: effects on extracellular matrix synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients.

Endothelin-1 (ET-1) seems to enhance the pro-fibrotic protein synthesis by skin fibroblasts and its effects are mediated by endothelin-A and B (ETA and ETB) receptors. This study aimed to investigate the effects of ETA and ETB receptor antagonists (ETARA-sitaxsentan and ETA/BRA-bosentan) on type I collagen (COL-1), fibronectin (FN) and fibrillin-1 (FBL-1) synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients. Primary cultures of fibroblasts were obtained from skin biopsies of 6 female systemic sclerosis patients and were treated with ET-1 (100 nM) for 24 and 48 hrs with or without pre-treatment (1 hr) with ETARA (2 µM) or ETA/BRA (10 µM). Primary culture of human scleroderma skin fibroblasts not treated with ET-1 or ET receptor antagonists (ETARA and ETA/BRA) were used as controls. COL-1, FN and FBL-1 synthesis was evaluated by immunocytochemistry and Western blot analysis. Immunocytochemistry and Western blot analysis showed that ET-1 significantly increased COL-1 and FN synthesis at 24 and 48 hrs and FBL-1 synthesis at 48 hrs vs untreated cells. ETARA significantly contrasted the ET-1-mediated increase in COL-1 and FN at 24 hrs as well as COL-1 and FBL-1 at 48 hrs, but not FN synthesis vs ET-1-treated fibroblasts. Conversely, ETA/BRA significantly antagonized the ET-1-mediated overproduction of COL-1 and FN both at 24 and 48 hrs and the FBL-1 synthesis at 48 hrs vs ET-1-treated cells. The single ETARA treatment seems to contrast significantly the increase in COL-1 synthesis, whereas the dual ETA/BRA treatment seems active in significantly antagonizing both COL-1 and FN overproduction induced by ET-1. In conclusion, ET-1 antagonism might have positive effects in contrasting the profibrotic activity of systemic sclerosis skin fibroblasts.

Anterior approach to the spine. Role of the general surgeon, techniques and surgical complications. The 10-year experience of the Niguarda Hospitals.

AIM: The aim of the study was to present the incidence of early and late surgical complications in a group of patients treated with anterior approach for spine lesions. The study was also focused on technical aspects for lesions of D1-D2 and L5-S1 segments and results on a group of patients underwent adjunctive surgical procedures at the same time of spinal surgery. METHODS: This was a retrospective study based on our database from April 1998 to December 2008. The study enrolled 120 consecutive patients (M/F 73/47; mean age 43.1 years; range 15-70 years) who underwent spinal surgery for trauma (92 patients), primitive or metastatic cancer (12 patients), benign lesion (2 patients), degenerative disc disease (6 patients) and infection disease (8 patients). This work describes the anterior approach to the spine. RESULTS: No death was recorded. Thirty-two patients (26.6%) presented postoperative complications: persistent urinary tract infections in 19 (15.9%), pneumonia in 6 (5%), pleural effusion in 3 (2.5%), wound infection in 2 (1.6%), retro-peritoneal abscess in 1 (0.8%) and haemorrhage in 1 (0.8%). During the follow-up (mean 3.8 years; range 2 months-10 years) 1 patient (0.8%) required two further surgical procedures for tuberculosis abscess recurrences. CONCLUSION: Anterior approach to the spine is effective and safe. Surgical complications do not negatively affect patient survival and spine stabilization included patients with D1-D2 and L5-S1 lesion and patients who receive adjunctive surgery at the same time of spine procedure.

[CTLA4-Ig interferes and downregulates the proinflammatory activities of rheumatoid synovial macrophages in monoculture]. / CTLA4-Ig influenza e inibisce le attività proinfiammatorie di macrofagi sinoviali reumatoidi in monocoltura.

OBJECTIVE: CTLA4-Ig, a biologic agent employed in rheumatoid arthritis (RA) treatment, downregulates the immune response and exerts anti-inflammatory effects acting on different cells including dendritic/T cells interaction and directly on osteoclasts. We investigated the anti-inflammatory effects of CTLA4-Ig in primary monocultures of RA synovial macrophages (SM). METHODS: SM were obtained, from 8 RA patients (7 F, 1 M; DAS28>5.2) who underwent therapeutic arthroscopic synoviectomy and were cultured in the absence and in the presence of CTLA4-Ig at the concentration of [500 microg/ml], the most reliable dose related to the previous pharmacological clinical and experimental experiences. Inflammatory cytokine (IL-6, TNFalpha, IL-1beta) expression was evaluated by immunocytochemistry (ICC with relative image analysis), western blot (WB), and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: ICC analysis revealed that CTLA4-Ig treatment significantly downregulated cytokine expression (p<0.001 for IL-6, TNFalpha and IL-1beta) when compared to untreated RA SM. WB and qRT-PCR confirmed partially the data. CONCLUSIONS: CTLA4-Ig was found to exert a direct and significant anti-inflammatory effect on primary monocultures of RA SM, suggesting a therapeutic power in different phases of the disease activity.

Endothelin and sex hormones modulate the fibronectin synthesis by cultured human skin scleroderma fibroblasts.

OBJECTIVE: To evaluate the influence of endothelin-1 (ET-1) and sex hormones on cell proliferation and extracellular matrix (ECM) synthesis (ie, fibronectin, laminin) by cultured normal and scleroderma (SSc) human skin fibroblasts (FBs). METHODS: Primary cultures of FBs were treated with ET-1 and sex hormones (17beta-oestradiol or testosterone) for 24 h. Cell growth was analysed by methiltetrazolium salt test, ECM synthesis was evaluated by immunocytochemistry and western blot, both at 24 h. RESULTS: In normal FBs, ET-1 and 17beta-oestradiol, as well as their combination, increased cell growth (p<0.001, p<0.001, p<0.01 vs untreated cells (control), respectively) and fibronectin synthesis (p<0.05, p<0.05, p<0.01 vs control, respectively). By contrast, testosterone either alone or in combination with ET-1 did not influence cell proliferation, but decreased fibronectin synthesis (p<0.05, testosterone vs control). In SSc FBs, ET-1 and 17beta-oestradiol alone or their combination induced an increased fibronectin synthesis (p<0.05, p<0.05, p<0.01 vs control, respectively). Unexpectedly, testosterone induced an increase of fibronectin synthesis (p<0.05 vs control). CONCLUSIONS: ET-1 and 17beta-oestradiol seem to exert a profibrotic effect in normal and SSc culture FBs and might suggest their synergistic effect in the pathogenesis of the fibrotic process in SSc.

Hydroxylated estrogen metabolites influence the proliferation of cultured human monocytes: possible role in synovial tissue hyperplasia.

INTRODUCTION: 17Beta-estradiol, estrone, and several of their hydroxylated metabolites, have been found to be significantly increased in synovial fluid of rheumatoid arthritis (RA) patients. In this study, we investigated whether the estrogen metabolites are able to exert direct effects on monocyte cell proliferation, which is important in RA synovial tissue activation and growth. METHODS: Human monocytes (THP-1) were treated with the following estrogen metabolites at different concentrations (from 10-8M, 10-9M, 10-10M to 10-11M) for 24, 48 and 72 hours: 16-hydroxyestrone (16OH-E1), 16-hydroxyestradiol (16OH-E2), 4-hydroxyestrone (4OH-E1), 4-hydroxyestradiol (4OH-E2), 2-hydroxyestrone (2OH-E1) and 2-hydroxyestradiol (2OH-E2). Monocytes were activated with interferon-gamma (INF-gamma). Cell cultures were also performed in presence of tamoxifen (10-7M) to evaluate whether the estrogen metabolites act through the estrogen receptors (ER). Cell growth was detected by MTT test and cell viability through the LDH release assay. RESULTS: 4OH-E1 and 2OH-E1 significantly increased cell growth at low concentration (10-10M), whereas they significantly reduced cell proliferation at high concentrations (10-9M). 16OH-E2 and 4OH-E2 induced opposite effects: cell proliferation at high concentration and antiproliferative action at low doses. On the contrary, 16OH-E1 and 2OH-E2 were found to be estrogen metabolites that induced cell proliferative effects for most of the tested doses. Tamoxifen caused the loss of effects on cell proliferation for almost all the metabolites. CONCLUSION: This study first demonstrates that different downstream estrogen metabolites interfere with monocyte proliferation and generally might modulate the immune response. Therefore, since estrogen metabolite/ratios are altered in the synovial fluid of RA patients, they might play important roles at least in RA synovial tissue hyperplasia.

Renal grafts from elderly donors: histological studies and long-term results.

To satisfy the increasing requests for renal grafts, elderly donors are increasingly accepted for kidney transplant at many centers. The main unresolved question is the long-term effect on graft survival of potential histological lesions due to donor age. We present a prospective histological study performed from January 1997 to December 2001 on 184 consecutively transplanted renal grafts in which the only criterion for graft acceptance was a normal value of serum creatinine upon admission to the intensive care unit independent of donor age. At the end of the study, 57 recipients (31%) of mean age 55 years (range 39 to 67 years) received a renal graft from donors aged more than 60 years (mean age 66 years; range 60 to 75 years), this cohort denoted as older donor kidney transplant group (ODKTG) and 127 recipients (69%) with a mean age of 49 years (range 21 to 63 years) received a renal graft from donors whose age was lower than 60 years (mean age 49 years; range 16 to 59 years), a cohort denoted as the younger donor kidney transplant group (YDKTG). The two groups were comparable for time of dialysis, cold ischemia time, immunosuppression therapy, grading of histological damage. At the end of the study with a mean follow-up of 5.6 years (range 3.5 to 7.5 years), primary graft nonfunction and delayed graft function were significantly more represented in the ODKTG than the YDKTG. Cumulative patient and graft survival was 84.3% and 79.4% in the ODKTG, respectively, and 93.8% and 85.9% in the YDKTG, respectively (P = NS). Cumulative serum creatinine values were 1.98 mg/100 mL in ODKTG and 1.65 mg/100 mL in YDKTG (P = NS). In conclusion, renal grafts from older donors presented histological damage comparable to that seen among renal grafts from younger donors.

Early and late residual renal function and surgical complications in living donors: a 15-year experience at a single institution.

Living donation in the field of renal transplantation has increased over time as well as the use of laparoscopic nephrectomy. We present a 15-year experience on 162 living donors (105 women, 57 men; mean age, 46.7 years; range, 31-74 years) who underwent nephrectomy using different surgical approaches as open lombotomic nephrectomy (OLN), open transperitoneal nephrectomy (OTN), and laparoscopic hand-assisted nephrectomy (LHAN). We collected data on residual donor and recipient renal function, as well as early versus late medical and surgical complications. With a mean follow-up of about 8 years, we observed normal residual renal function in all donors and similar results of early and late graft function independent of the surgical procedure. Long-term incidence of hypertension and noninsulin-dependent diabetes in living donors was similar to the general population. OLN and OTN donors showed higher incidences of early and late complications, readmissions, and reoperations than LHAN donors. Our results confirmed that living donor nephrectomy is a safe procedure without serious side effects in terms of renal function and long-term quality of life. LHAN should be the preferred technique because of a lower incidence of early and late complications.

Pancreas retransplantation: ideal timing and early and late results.

Surgical complications are the leading cause of pancreatic graft loss among diabetic patients who undergo pancreas transplantation alone (PTA), or combined with kidney transplantations (PK) or after kidney transplantations (PAK). Therapeutic effects on secondary complications of diabetes justify pancreas retransplantation (re-PT) when the first graft is lost. However, the appropriate timing for retransplant and related problems is not known. We present our initial experience on re-PT performed on seven diabetic patients who lost their first pancreas grafts (PK) due to surgical complications (venous thrombosis in five and enteric fistula in two). Five re-PT were performed a few days after the first PT without a second course of induction therapy, while two patients received re-PT some months later with reinduction therapy. In the early re-PT group, one patient died some hours after the second surgical procedure due to pulmonary embolism, while four patients lost their second grafts due to accelerated rejection within 2 years from re-PT. In the late re-PT group, both patients have good graft function without signs of rejection. Our initial experience showed discouraging results in the group of early re-PT, due to accelerated rejection episodes leading to a high incidence of graft loss. Late re-PT accompanied by reinduction therapy seemed to have better results.

Laparosopic hand-assisted living donor nephrectomy: the Niguarda experience.

Perioperative donor morbidity, a barrier to living organ donation, may be mitigated by the laparoscopic approach. From September 2002 to September 2004, 15 living donors, of ages ranging from 36 to 59 years, underwent laparoscopic nephrectomy. We used a hand-assisted device to increase the safety of the procedure. The average operating time was 200 minutes. The average blood loss was about 100 mL. The patients resumed oral intake and started walking within 1 day. The average postoperative hospital stay was 6 days. Although laparoscopic operating times were longer than those for traditional surgery, we showed benefits to the laparoscopic donor to be less postoperative pain, better cosmesis, shorter recovery time, and faster return to normal activities. We therefore consider laparoscopic nephrectomy a good alternative to traditional surgery for selected patients. Despite a lack of strong evidence, such as large prospective randomized studies, laparoscopic donor nephrectomy is likely to become the gold standard for donor nephrectomy in the near future.

Advantages of short-time ureteric stenting for prevention of urological complications in kidney transplantation: an 18-year experience.

We retrospectively studied the incidence of urological complications in a consecutive series of 590 patients (group B) who received a kidney transplant (KT) with a ureteral stent from January 1994 to December 2002. The ureteral stent was sewn to the bladder catheter during the surgical procedure and left in situ for a mean time of 10 days (range 8 to 12 days). The results were compared to a consecutive series of 414 patients who received a KT from March 1986 to December 1993 without a ureteral stent (group A). The two groups were comparable in terms of donor and recipient gender, ischemia time, delayed graft function, and chronic rejection incidence, but differed in mean donor age (44.1 vs 36.0 years), mean recipient age (45.4 vs 39.1 years), living/cadaveric donor rate (19.8% vs 11.9%), arterial lesions and bench reconstruction rate (11.1 vs 3.5%), as well as acute rejection episodes (11.7% vs 29.2%). Complications were seen in nine patients in group B (1.5%) and 17 patients in group A (4.1%) (P < .0001). Urinary leaks presented in two patients in group B (0.3%) and 11 patients in Group A (2.6%; P < .0001), while stenosis was present in six patients in group B (1.5%) and 7 in group A (1.2%) (P = NS). Urological complications such as urinary tract infection and macroscopic hematuria were similar in both groups. Time to presentation of a leak was within 2 weeks from KT in 10 patients (92.3%), while stenosis presented early in four patients (one in group B and four in group A). Of the stenoses, 69.3% presented late (beyond 12 weeks) in five patients in group B and three in Group A. In conclusion, our data suggest that routine use of double pigtail ureteral stent significantly decreased the incidence of leaks and early stenoses, but it did not modify late stenosis incidence. In the last decade, risk factors for urological complications have been increasing over time, namely, older donors and older recipients, living donation, length of dialysis, and the use of grafts with arterial lesions. Therefore we believe that a ureteral stent should be routinely considered to afford the advantage to protect the urinary anastomosis in the early postoperative period when the incidence of complications is highest, without the need of cystoscopy for its removal.

Surgical complications are the main cause of pancreatic allograft loss in pancreas-kidney transplant recipients.

We examined surgical complications among a group of diabetic type 1 patients (IDDM) with end-stage renal disease (ESRD) who had undergone pancreas-kidney transplantations (PK). Between October 1993 and August 2004, 70 SPK were performed using bladder (n = 14) or enteric (n = 56) drainage. Donors were selected according to standard criteria (mean age, 27.6 years; range, 17-49). All patients received cyclosporine-based immunosuppression. All pancreata functioned immediately, whereas 2 patients needed postoperative dialysis. Four patients (5.7%) lost their pancreatic graft due to vascular thrombosis; both patients underwent urgent allograft pancreaectomy and pancreas retransplantation (re-PT). One of them (1.4%) experienced a venous thrombosis and died due to a pulmonary embolism at 12 hours after re-PT. The other 3 patients had uneventful postoperative courses and were discharged with good pancreatic and renal function. Three patients in the bladder group (21.4%) had an anastomotic leak, which resolved with a bladder catheter. Four patients in the enteric group (7.1%) who experienced an anastomotic leak needed a second surgical procedure but in 3 of them allograft pancreatectomy was necessary. Relaparotomy was required in the other 3 patients due to hemorrhage (1 patient) or occlusion (2 patients). Acute rejection episodes, which occurred in 16 patients (22.8%), were treated with steroid boluses. With a mean follow-up of 72 months (range, 3-129), 2 patients have died at 8 and at 36 months, respectively, after SPK due to acute myocardial infarction (2.9%). Chronic rejection was the leading cause of pancreatic failure in 5 patients (7.1%) and of renal failure in 2 patients (2.8%). Patient, kidney, and pancreas survival rates were 95.8%, 92.9%, and 81.5%, respectively. Surgical complications were the leading cause of pancreatic allograft loss in IDDM and ESRD patients submitted to SPK.

Drinking habits and performance in an attention test in young people frequenting discotheques.

Throughout July and August 2002, at the same time as a campaign on driving safety, questionnaire and breath alcohol tests was submitted to young people outside the best-frequented discotheques of the Apulia area (Italy), to describe the role of alcohol intake in the evening-time amusement and assess its influence on their attention level. 217 young people, aged between 18-30 years, accepted to fill in a questionnaire, and undergo a 'breathalizer' test and attention test, before and after having spent time in the discotheque. The score difference between entry and exit on the attention test was -1.48 points and alcohol blood level increase up to 0.14 g/l. Established the lower reference limit for the attention test to be 21 points, it was found that 9.7% had an attention score lower than the reference limit, and 61.9% of this group had a blood alcohol level higher than 0.5 g/l. A linear regression model between the difference of blood alcohol level before and after the time spent in the discotheque and attention score on exiting from it, turned out to be statistically significant. It's necessary to go on with continuous police checks and informative awareness campaigns regarding alcohol use, driving and night amusement.

[The discharge form: advantages and limits legionellosis cases individuation]. / La Scheda di Dimissione Ospedaliera: vantaggi e limiti nell'individuazione dei casi di legionellosi.

Despite legionellosis surveillance is active in Italy since many years, the disease notification appears still undervalued. A multicentric survey was carried out among 5 big Italian hospitals. It examined 11,435 discharge forms (1999-2001), reporting pneumonia diagnosis. Legionellosis (II class of notify system for infectious disease) was studied among pneumonia diagnosis by discharge forms. According to the ICD9-CM, there's no specific code for legionellosis (this disease is included among "others gram-negative pneumonia"). So the presumed pneumonia imputable to Legionella spp were the 2.7% of the whole number of analyzed discharge forms. Besides, the data regarding the other pneumonia showed that the etiological agent was specified only in the 11.2% of the case. This situation could be rectified both introducing adequate discharge forms codes and promoting the etiological diagnosis during the hospital stay.

Acute myositis following autologus bone marrow transplantation (BMT).

A 4-year-old boy with acute myeloid leukemia developed acute myositis associated with refractory thrombocytopenia one month after autologous bone marrow transplantation (BMT). Clinical, electromyographic and biohumoral features were consistent with the diagnosis of myositis. The patient responded to corticosteroids, and 39 months after BMT he is in complete remission and has regained good muscle function. Although we could not determine with certainty the specific pathophysiologic mechanism of this complication, it should be pointed out that acute myositis can occur in the early post-BMT period.

[Use of cyclosporin and verapamil in association with chemotherapy in the treatment of pediatric patients with advanced-stage neoplasms. A pilot study]. / Impiego della ciclosporina e del verapamile in associazione a chemioterapici nel trattamento di pazienti pediatrici con neoplasia in fase avanzata. Studio pilota.

Multidrug resistance represents one of the most important factors that may lead to a therapeutic failure in some patients affected by malignancies. One of the best known mechanisms is linked to the genic amplification or the overproduction of a membrane glycoprotein, GP170, that is the product of the gene MDR1. The existence of drugs (calcium blockers, cyclosporine, tamoxifen, reserpine, quinidine) able to bind themselves to gp170 and to paralyze its activity in vitro is well known. We studied 20 pediatric patients (median age 9 years) affected by acute lymphoblastic leukemia (ALL), osteosarcoma, neuroblastoma and medulloblastoma, in advanced stage of disease. We employed in all cases the association of cytostatics with verapamil (50-70 mg/m2 i.v.) and cyclosporine (5-8 mg/kg i.v.) with different infusion schedules. In leukemias we administered vincristine (1.5 mg/m2), and daunomycin (40 mg/m2), in solid tumors VP16 (150 mg/m2) and adriamycin (60 mg/m2). Seventy-two therapeutic courses were performed: 39 in ALL, 16 in osteosarcoma, 16 in neuroblastoma and 1 in medulloblastoma. On the whole 5 complete remissions were achieved in ALL patients and 1 in an osteosarcoma patient. We did not observe a significant myelosuppression during treatment, therefore few infectious complications occurred; furthermore electrocardiographic changes have been mild and promptly resolved after temporary discontinuation of verapamil infusion. Our data suggest a synergy of verapamil and cyclosporine in the inhibition of multidrug resistance induced by gp170, without the occurrence of heavy toxicity. The results obtained in ALL patients are encouraging., especially in view of a possible subsequent bone marrow transplantation, while in solid tumors they are not as satisfying.

Post-extubation non-cardiogenic pulmonary edema.

Non-cardiogenic pulmonary edema (NCPE) is a rare entity manifested by pulmonary edema with normal pulmonary arterial pressures. NCPE has been described in children and recently reported in adults. Onset is rapid and late recognition or inappropriate treatment may lead to a fatal outcome. Most adult cases have been attributed to laryngospasm and generation of a large negative intrathoracic pressure causing transcapillary filtration. The majority of reported adult cases were at risk for Upper Airway Obstruction (UAO). This report describes three young, active duty men who developed this entity following routine uncomplicated surgical procedures. Intubations were uncomplicated and fluid management appropriate. No patient had risk factors for UAO. Early extubation with respiratory attempts against a closed glottis may be etiology for this complication.

Xenobiotic-metabolizing enzyme systems in test fish--IV. Comparative studies of liver microsomal and cytosolic hydrolases.

1. The initial slopes of the substrate-activity curves of several hydrolases were determined in the microsomal and cytosolic fractions of the liver of several fish recommended by OECD for the regulatory testing of chemicals. 2. Inter-species differences ranged within a factor of 7-17 for the esterases and reached a factor of 60 for the amidase. Guppy and carp appeared endowed with hydrolase activities which, overall, are much higher than zebra fish, trout and golden orfe. 3. The comparison with the rat liver microsomal hydrolases strongly suggests that fish are endowed with similar or higher levels of A-esterase and with much less B-esterase/amidase activities.

Long-term followup of penetrating abdominal aortic injuries after 15 years.

Eleven of 14 survivors who sustained trauma to the abdominal aorta have been evaluated 16 to 18 years after injury through personal interview, physical examination, and abdominal contrast computerized tomography (CCT). The average age of survivors was 39 years (range, 37-47). All patients had minimal debridement of the aortic injury with lateral arteriorrhaphy. No patients had symptoms of arterial insufficiency. However, five patients had abnormal ankle/brachial indices (ABI). In four patients, ABI was less than 1.00 at rest and a fifth patient's ABI decreased significantly: 0.60 left and 0.65 right from an average of 1.00 bilaterally after standardized exercise treadmill. CCT evaluation revealed aortic calcification in five patients in the area of aortic injury. Aortic calcification occurred only in the patients with abnormal ABI's. This long-term followup identifies no evidence for late compromise in the aorta; however, there is a suggestion that injury and repair may contribute to the accelerated development of atherosclerosis.