Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT), which catalyzes conversion of glyoxylate into glycine. AGT deficiency results in overproduction of oxalate that ultimately leads to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only available therapy to prevent disease progression. Gene therapy is an attractive option to provide an alternative treatment for PH1. Toward this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. Compared with saline controls, AGT-deficient mice injected with an HDAd encoding the AGT under the control of a liver-specific promoter showed a significant reduction of hyperoxaluria and less increase of urinary oxalate following challenge with ethylene glycol, a precursor of glyoxylate. These studies may thus pave the way to clinical application of HDAd for PH1 gene therapy.

SR-A and SREC-I binding peptides increase HDAd-mediated liver transduction.

Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes without inducing chronic toxicity. However, vector therapeutic index is narrow because of a toxic acute response with potentially lethal consequences elicited by high vector doses. Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) are major barriers to efficient hepatocyte transduction. We investigated two small peptides (PP1 and PP2) developed by phage display to block scavenger receptor type A (SR-A) and scavenger receptor expressed on endothelial cells type I (SREC-I), respectively, for enhancement of HDAd-mediated hepatocyte transduction efficiency. Pre-incubation of J774A.1 macrophages with either PP1 or PP2 prior to HDAd infection significantly reduced viral vector uptake. In vivo, fluorochrome-conjugated PP1 and PP2 injected intravenously into mice co-localized with both CD68 and CD31 on KCs and LSECs, respectively. Compared with saline pre-treated animals, intravenous injections of both peptides prior to the injection of an HDAd resulted in up to 3.7- and 2.9-fold increase of hepatic transgene expression with PP1 and PP2, respectively. In addition to greater hepatocyte transduction, compared with control saline injected mice, pre-treatment with either peptide resulted in no increased levels of serum interleukin-6, the major marker of adenoviral vector acute toxicity. In summary, we developed small peptides that significantly increase hepatocyte transduction efficacy and improve HDAd therapeutic index with potential for clinical applications.

Retinal transduction profiles by high-capacity viral vectors.

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, the limited cargo capacity of AAV prevents their use for therapy of those inherited retinopathies (IRs) due to mutations in large (>5 kb) genes. Viral vectors derived from adenovirus (Ad), lentivirus (LV) and herpes virus (HV) can package large DNA sequences, but do not target efficiently retinal photoreceptors (PRs) where the majority of genes responsible for IRs are expressed. Here, we have evaluated the mouse retinal transduction profiles of vectors derived from 16 different Ad serotypes, 7 LV pseudotypes and from a bovine HV. Most of the vectors tested transduced efficiently the retinal pigment epithelium. We found that LV-GP64 tends to transduce more PRs than the canonical LV-VSVG, albeit this was restricted to a narrow region. We observed more extensive PR transduction with HdAd1, 2 and 5/F35++ than with LV, although none of them outperformed the canonical HdAd5 or matched the extension of PR transduction achieved with AAV2/8.

Recurrent myocardial ischaemia during combination antiviral therapy in a patient with chronic hepatitis C and normal aminotransferase levels.

A 63-year-old man with chronic hepatitis C and persistently normal alanine aminotransferase levels was treated with peginterferon alpha-2a (180 microg weekly) and ribavirin (800 mg daily). Hepatitis C virus-ribonucleic acid was negative at week 4. After 12 weeks of therapy, haemoglobin levels had decreased by 3.5mg/dL, and he developed a syncope episode with electrocardiographic signs of myocardial ischaemia. Antiviral treatment was stopped and the ischaemia-like electrocardiographic changes resolved completely within 2 months. Eight months later, because of the previous rapid virological response and patient motivation, he was treated again with peginterferon and ribavirin. Baseline and weekly electrocardiographic recordings were obtained during treatment. At week 4 hepatitis C virus-ribonucleic acid was negative. At week 8, when the haemoglobin levels had decreased by 3.4 mg/dL, the patient developed the same ischaemia-like changes that occurred during the previous treatment. Antiviral therapy was stopped and the electrocardiographic ischaemia-like changes disappeared after 1 month. The patient neither had a history of previous cardiovascular diseases, nor evidence of current disease at myocardial scintigraphy. However, a coronary microvessel spasm, possibly related to drug-toxicity and/or anaemia could not be excluded. This case indicates the need of strict electrocardiographic monitoring in elderly patients undergoing treatment with peginterferon and ribavirin.

Escharotomies, fasciotomies and carpal tunnel release in burn patients--review of the literature and presentation of an algorithm for surgical decision making.

Escharotomies are usually performed in patients with circumferential third degree burns of the extremities or anterior trunk. Fasciotomies are recommended for patients who sustained high voltage (or associated crush) injuries, with entrance or exit wounds in one or more extremities. Carpal tunnel release is practiced routinely in some services for cases of electrical injury. We have reviewed the literature which provides relatively little information as to when should these procedures actually be performed and what would happen if they were not done. We present a series of patients treated at our institution when an algorithm was used for surgical decision making as to when (or not) to operate (perform an escharotomy, a fasciotomy or a carpal tunnel release), based on clinical signs and monitoring alternatives, using the oximeter and the Doppler flowmeter. 13 938 burn patients were treated at our institution during the year of 2005. Of these, 571, with an average of 22.3 % TBSA, were treated as inpatients. Of these, 58 (10.3 %) had circumferential or electrical burns of one or more extremities. Patients were monitored hourly from admission and decision to operate was based on clinical signs and in absent or below 90 % oximetry, regardless of Doppler flow signs. 68 % were males, 6 (11.3 %) patients had immediate escharotomies, while 4 (7.5 %) had immediate fasciotomies. 2 of these patients were operated regardless of positive Doppler sign but no oximetry. All patients recovered oximetry over 90 % immediately after the operations. 3 patients had negative Doppler sign but oximetry > 90 % and were not operated. 3 patients had carpal tunnel releases based on oximetry < 90 % and symptoms of compression of the median nerve. Patients who were not operated fared well with no signs or symptoms of impairment of circulation or nerve damage up to their 3 and 6 months reevaluations.

EP2 receptor stimulation promotes calcium responses in astrocytes via activation of the adenylyl cyclase pathway.

Astrocytes are a heterogeneous population of cells that are endowed with a great variety of receptors for neurotransmitters and neuromodulators. Recently prostaglandin E2 has attracted great interest since it is not only released by astrocytes but also activates receptors coupled to either phospholipase C or adenylyl cyclase. We report that EP2 receptor stimulation triggers cAMP production but also causes release of Ca2+ from intracellular stores. This effect is shared by other receptors similarly coupled to adenylyl cyclase and elicited by direct stimulation of the enzyme or application of cAMP analogues. However, the stimulation of the Ca2+ response by cAMP is not mediated by protein kinase A, since a specific antagonist of this kinase had no effect. Such a cross-talk between cAMP and Ca2+ was not observed in all astrocytes. It might therefore reflect a specific resource of either a subpopulation or astrocytes in a specific functional state.

Avoiding axillary dissection in breast cancer surgery: a randomized trial to assess the role of axillary radiotherapy.

BACKGROUND: The need to dissect axillary nodes in patients with early breast cancer and clinically negative axilla remains controversial. The aim of the study was to assess the role of axillary radiotherapy (RT) in reducing axillary metastases in patients with early breast cancer who did not receive axillary dissection. PATIENTS AND METHODS: From 1995 to 1998, 435 patients over 45 years old with breast cancer up to 1.2 cm and no palpable axillary nodes were randomized 214 to breast conservation without axillary treatment and 221 to breast conservation plus axillary RT. RESULTS: After a median follow-up of 63 months, overt axillary metastases were fewer than expected: three cases in the no axillary treatment group (1.5%) and one in the RT group (0.5%). Expected cases were 43 in the no axillary treatment group and 10 in the RT group. Rates of distant metastases and local failures were low, and 5-year disease free survival was 96.0% (95% confidence interval, 94.1%-97.9%) without significant differences between the two arms. CONCLUSIONS: This study suggests that occult axillary metastases might never become clinically overt and axillary dissection might be avoided in patients with small carcinomas and a clinically negative axilla. Axillary RT seems to protect the patients from axillary recurrence almost completely.

Dendritic targeting of mRNAs for plasticity genes in experimental models of temporal lobe epilepsy.

PURPOSE: To analyze whether the subcellular localization of the messenger RNAs (mRNAs) coding for the neurotrophin brain-derived neurotrophic factor (BDNF), its receptor TrkB, and the alpha and beta subunits of calcium-calmodulin-dependent kinase II (CaMKII) are modified after pilocarpine and kindled seizures. METHODS: Epilepsy models: pilocarpine and kindling. Analysis of mRNA levels in the dendrites: high-resolution, nonradioactive in situ hybridization. RESULTS: Nonstimulated rats: BDNF, TrkB, and CaMKII-beta mRNAs localized in the soma and in the proximal dendrites of hippocampal pyramidal cells, and in the soma only of dentate gyrus (DG) granule cells; CaMKII-alpha mRNA localized throughout the dendritic length in neurons of all hippocampal subfields. Pilocarpine seizures: increased staining levels of CaMKII-alpha mRNA throughout the whole dendritic length in all hippocampal subfields; induction of CaMKII-beta, BDNF, and TrkB mRNAs dendritic targeting in CA1, CA3, and DG neurons. Class 2 kindled seizures: increase in dendritic staining intensity for CaMKII-alpha in CA1, CA3, and DG neurons; induction of dendritic localization of CaMKII-beta, BDNF, and TrkB mRNAs in CA3 neurons. Fully kindled seizures: no change in the subcellular distribution of BDNF, TrkB and CaMKII-beta mRNAs; reduction of CaMKII-alpha mRNA dendritic staining, as compared with unstimulated kindled animals. CONCLUSIONS: Data provide evidence that BDNF, TrkB, and CaMKII-alpha and -beta mRNAs are accumulated in the dendrites of specific hippocampal neurons during pilocarpine seizures and kindling development. The dendritic targeting of these genes may be causally involved in epileptogenesis and thus may represent a new therapeutic target for some forms of partial epilepsy.

Acetylphenylhydrazine induced haemoglobin oxidation in erythrocytes studied by Mössbauer spectroscopy.

The oxidative action of acetylphenylhydrazine (APH) on red blood cells obtained from healthy donors and from patients with breast cancer has been investigated by Mössbauer spectroscopy. Whole blood was incubated with APH for different time periods and the Mössbauer spectra of the packed red cells were recorded and compared. The evolution with time of the oxidation products has been followed. The largest difference in red cells analysis between healthy persons and patients was found after about 50 min of treatment where Mössbauer spectra of patient samples show a much broader spectral pattern due to an advanced haemoglobin oxidation.

Aortic graft in the jejunum without bleeding. A real surprise at endoscopy.

It is known that prosthetic infection, graft-duodenal fistula, and erosion are possible late complications after aortic reconstruction, and that all these reported complications are accompanied generally by variable bleeding with different presentations. We report the case of a 63-year-old man who underwent a diagnostic upper gastrointestinal endoscopy for investigation of nausea, anorexia, asthenia, fever, and mild leukocytosis. The patient's medical history included a gastric resection for ulcer, with Billroth II gastrojejunostomy reconstruction and implantation of a Dacron vascular graft for abdominal aortic aneurysm 20 years and 3 years earlier, respectively. Abdomen ultrasonography showed hypoechoic area around an aortic prosthesis. Endoscopy found a foreign body corresponding to the vascular graft at the jejunum. No signs of bleeding were recorded. The patient was hospitalized and submitted to surgery that involved extra-anatomic axillofemoral bypass, bowel resection with a gastrojejunum Roux anastomosis, and prosthesis removal.

C-reactive protein is increased in patients with degenerative aortic valvular stenosis.

OBJECTIVES: The goal of this study was to assess the presence of systemic inflammation in degenerative aortic valvular stenosis. BACKGROUND: Local inflammatory changes, resembling those observed in atherosclerosis, have been recently reported in degenerative aortic valvular stenosis. It is presently unknown whether systemic signs of inflammation, similar to those observed in atherosclerosis, may be present in this disorder. METHODS: C-reactive protein (CRP) was measured by enzyme immunoassay in 141 subjects: 62 with trileaflet degenerative valvular aortic stenosis and 79 volunteers with similar demographic and clinical characteristics. IgG antibodies against Helicobacter pylori (enzyme-linked immunosorbant assay) and Chlamydia pneumoniae (microimmunofluorescence assay) were also measured. RESULTS: C-reactive protein levels (mg/dl, mean +/- SD) were 0.848 +/- 1.42 in patients and 0.394 +/- 0.50 in controls (p = 0.0001, Mann-Whitney U test). Seroprevalence of H. pylori was 68.7% in patients and 79.7% in controls (p = NS), whereas seroprevalence of C. pneumoniae infection was higher in patients than it was in controls (59.7% vs. 33%, p = 0.003; chi-square test). After adjustment for various covariates in multiple logistic regression, the odds ratio for degenerative aortic stenosis was 3.41 for C. pneumoniae infection (95% confidence intervals [CI]: 1.60 to 7.30) and 2.76 for CRP (95% CI: 1.08 to 7.05). There was no significant difference in patients or controls in CRP levels according to the serostatus for C. pneumoniae. CONCLUSIONS: Systemic signs of inflammation, similar to those found in atherosclerosis, are present in patients with degenerative aortic valve stenosis. They do not seem to be linked to C. pneumoniae or H. pylori infection.

Anticoagulant and antiprotease profiles of a novel natural heparinomimetic mannopentaose phosphate sulfate (PI-88).

Heparinomimetic mannopentaose phosphate sulfate (PI-88) (Progen Industries Ltd. Brisbane, Australia), currently developed as an anticoagulant and antiproliferative agent, mainly is composed of a pentomannan. However, tetrasaccharide and disaccharide components are also present. The molecular profile and the anticoagulant potency of PI-88 are investigated in this study. Gel permeation chromatography and polyacrylamide gel electrophoresis analyses were carried out to determine the molecular profile and separation of components of PI-88, respectively. Potentiation of antithrombin III (ATIII) and heparin cofactor-II (HC-II) activity were measured using chromogenic substrate assay. In order to determine anticoagulant and antiprotease effects of PI-88, various global anticoagulant tests, such as the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Hep-test (Haemachem Inc., St. Louis), ecarin clotting time (ECT), activated clotting time (ACT), and thromboelastography (TEG) were used. Anti-Xa and anti-IIa activities also were measured. The effect of PI-88 on the release of tissue factor pathway inhibitor (TFPI) was performed in nonhuman primates who received PI-88 and in endothelial cell culture systems. The relative susceptibility of PI-88 to heparinase I, protamine sulfate (PS), and platelet factor 4 (PF4) also was evaluated. The high-performance liquid chromatography profiles of PI-88 showed that its average molecular weight is approximately 2300 Da. Separation and gradient electrophoretic patterns of PI-88 showed that it is composed of five different fractions. This agent activates HC-II through inhibiting the thrombin generation but not inhibiting ATIII. Although PI-88 produced a concentration-dependent prolongation of all of the clotting tests, ECT gave the best correlation in the dose-response curve (ECT, r2 = 0.94; TT, r2 = 0.84; APTT, r2 = 0.69). Heparinomimetic mannopentaose phosphate sulfate (PI-88) exhibited marked inhibition of FIIa, but not of FXa. Heparinase I failed to produce significant neutralization of PI-88 in all the assays used, whereas PS and PF4 partially neutralized the effects of this compound. Heparinomimetic mannopentaose phosphate sulfate (PI-88) produced fivefold increase in the TFPI levels at 15 minutes after intravenous (IV) injection to primates. The incubation of PI-88 in endothelial cell culture system also showed a strong effect on TFPI release. These results suggest that PI-88 exhibited strong antithrombotic and anticoagulant activity in addition to its known antiproliferative properties. Because of the molecular characteristics and the dual nature of the pharmacologic action of PI-88, this agent represents an attractive pharmacologic agent for the control of thrombotic and proliferative disorders.

Global anticoagulant effects of a novel sulfated pentomanan oligosaccharide mixture.

PI-88 is a potent antiproliferative agent, which is developed for various indications in cancer. This agent is obtained from yeast fermentation and is primarily composed of pentamannose and tetramannose oligosaccharide units. PI-88 is capable of producing anticoagulant effects, which are mediated by heparin cofactor II. The purpose of this study was to determine the anticoagulant properties of PI-88 in native whole blood, freshly drawn from human volunteers, supplemented with PI-88 at various concentrations (0-100 microg/mL). Whole blood activated clotting time (ACT) was measured using Hemochron instruments. PI-88 produced a strong anticoagulant effect at 100 microg/mL (479.0+/-59.5 sec). This anticoagulant effect was comparable to that observed in interventional cardiology and open-heart surgery. At the lower level, PI-88 produced concentration-dependent effects on ACT. Using thromboelastographic techniques (TEG), the effect of PI-88 was measured in terms of various parameters. PI-88 produced potent anticoagulant effects in the TEG studies. At the concentration of 25 microg/mL, it produced a complete anticoagulant effect in whole blood. Whole blood samples supplemented with PI-88 showed a concentration-dependent decrease in the generation of various markers of clotting activation. These results clearly suggest that PI-88 exerts an anticoagulant effect in whole blood. Because of the low-molecular-weight nature and a novel mechanism of action, this new drug may be considered for further development, particularly in cancer patients.

Endoscopic treatment of colorectal benign-appearing lesions 3 cm or larger: techniques and outcome.

PURPOSE: Colonoscopic polypectomy is the preferred technique to remove the majority of polyps. The authors evaluate feasibility, safety, and the effectiveness of endoscopic treatment of colorectal benign-appearing polyps equal to or larger than 3 cm. METHODS: Ninety-seven patients with 104 giant polyps underwent polypectomy within a nine-year period. The majority of these procedures were performed on an outpatient basis, all on unsedated patients. Gross appearance, size, location, histologic characteristics, synchronous lesions, modality, and adequacy of removal of giant polyps were analyzed. The follow-up was achieved in 89 percent of patients during a period ranging from 6 to 96 months (median, 38). RESULTS: Of the 104 removed polyps, 75 (72 percent) were adenomatous, 2 (2 percent) were hyperplastic, and 27 (26 percent) were malignant polyps. Six patients had more than one giant polyp. Several additional smaller polyps were found in 52 patients and a synchronous cancer in 4. Twenty-one (20 percent) giant polyps were equal to or larger than 4 cm. Forty-nine were pedunculated, 20 were short-stalked, and 35 were sessile. Sixty-one polyps were excised in one piece, and forty-three were excised using a piecemeal technique. Only four complications (3.8 percent) were recorded; all cases were treated endoscopically. Fifty-eight (75 percent) adenomas and eighteen (67 percent) malignant polyps were completely excised. Surgery was performed in 7 of 27 patients (27 percent) with malignant polyps, where there was a doubtful, infiltrated margin or poorly differentiated cancer. Postpolypectomy surveillance permitted the detection and treatment of 25 metachronous or recurrent polyps and a metachronous cancer. CONCLUSIONS: This study shows that polypectomy of giant colorectal polyps, performed by an expert endoscopist, is feasible, effective, and safe, even on an outpatient basis. The authors confirm that malignant polyps with incomplete excision, lymphovascular invasion, and poor differentiation require bowel resection. Postpolypectomy surveillance is useful for all patients who have undergone colonoscopic resection of giant adenomatous or malignant polyps.

Infection with Helicobacter pylori and leukocyte response in patients with myocardial infarction.

To test whether Helicobacter pylori may contribute to the inflammatory response following myocardial infarction, the levels of IgG antibodies to Helicobacter pylori and some parameters of leukocyte activity were measured in 63 patients and 61 comparable controls. Helicobacter pylori-positive patients showed a significantly higher expression of the adhesion molecule LFA-1 on neutrophils than Helicobacter pylori-negative patients (433+/-29.0 vs. 398.8+/-38.9 mean fluorescence channels; P<0.0001), whereas no significant difference for any parameters tested was found in control subjects. These data suggest a role of Helicobacter pylori in inducing a leukocyte response following myocardial infarction.

Large hyperplastic polyps of the colon.

Hyperplastic polyps are the most frequent nonneoplastic lesions of the colon. Typically, they are small sessile polyps (5 mm) located in the rectosigmoid area. Recently, they have been identified as markers of neoplastic polyps. Herein we describe four cases of large (20 mm in size) hyperplastic polyps found at our institution over a 9-year period. All four polyps were excised by endoscopic polypectomy on an outpatient basis without complications. Two polyps were in the right colon; one was pedunculated, none of them was associated with synchronous neoplastic polyps or polyposis. Up to now, follow-up in three patients has been negative for metachronous polyps. We conclude that a large hyperplastic polyp is an unexpected and rare finding, difficult to distinguish, and not related to particular colonic sites or synchronous adenomatous lesions. These polyps should be removed with a standard technique, and patients need to be followed with successive endoscopies.

[Biopsy of sentinel lymph nodes in the treatment of breast carcinoma: experience of the Surgery Department of the Hospital of Parma]. / La biopsia del linfonodo sentinella nel trattamento del carcinoma mammario: esperienza del Dipartimento Chirurgico dell'Azienda Ospedaliera di Parma.

Radical axillary nodes dissection in breast cancer is a standard for a correct staging, unfortunately this approach can cause several unpleasant sequelae and complications. Sentinel node biopsy applied to breast tumors could be a good option for predicting axillary nodes status avoiding complete dissection. The aim of this work is to report our experience with sentinel node biopsy during a period of 18 months. One hundred and nine patients with an infiltrating breast tumor T1 had been studied. There wasn't clinical and ultrasonographic evidence of axillary infiltration. Tumors had been injected on the day before surgery with a mixture of colloidal human albumin particles marked with 99m Technetium. In 108 out of 109 patients (99%) sentinel node had been identified using a gamma probe and biopsied during surgical intervention performed under local anesthesia. Sentinel node has been examined both with conventional histology and immunohistochemistry. In 26 cases the node was positive for metastases. Radical axillary dissection in this subgroup of patients showed that in 85% of them sentinel node was the only positive. We conclude that sentinel node biopsy can be a good alternative to traditional axillary dissection but there are still important questions about the best method of analysis and, before the technique become a routine procedure in breast cancer management, we should know the results of prospective clinical trials comparing survival of patients staged by sentinel node biopsy versus traditional axillary dissection.

Endoscopic preoperative colonic tattooing: a clinical and surgical complication.

Endoscopic colonic tattooing is the simplest and most economic technique for identifying small lesions or polypectomy sites during open and laparoscopic surgery. Moreover, it is useful for the endoscopic follow-up of polypectomy sites. India ink is the agent of choice because of its long-lasting stain and the low risk of adverse reaction and toxicity. Very few cases of complications have been reported. We report here the case of a patient in whom colonic tattooing in preparation for surgical resection was followed by clinical complications such as fever and abdominal pain. An abscess-type inflamed pseudotumor was found at laparotomy. Histological examination revealed chronic granulomatous inflammation.

Leukocyte count and aggregation during the evolution of cerebral ischemic injury.

We evaluated leukocyte aggregation by means of the leukergy test and count in 26 patients with atherothrombotic stroke and in 10 patients with transient ischemic attacks. The evaluation was performed within 24 h from the onset of symptoms and then repeated on day 2, 4, 6 and 8. Data were compared with those of 10 healthy controls. Stroke patients were followed until day 30 when a clinical examination and brain computed tomography were performed to evaluate the extent and outcome of cerebral damage. Both leukocyte aggregation and count were significantly increased in stroke patients with respect to controls. While leukocyte count was not able to differentiate the severity of neurological impairment in stroke patients, leukocyte aggregation was significantly higher in major than in minor stroke patients on days 2 and 4 (p < 0.05). Moreover, while values of leukocyte count recorded at entry remained substantially stable in the following determinations in all groups, leukocyte aggregation showed a significant increase (p < 0.05) on day 4 with respect to all the other determinations in major stroke patients. These findings show that the extent and temporal profile of changes in leukocyte count and aggregation are different in patients with cerebrovascular disease and suggest an involvement of altered leukocyte rheology in the development of cerebral ischemic injury.