The use of size-exclusion chromatography in the isolation of supercoiled minicircle DNA from Escherichia coli lysate.

Minicircle DNA (mcDNA) is the new cutting-edge technology which researchers have been exploring for gene therapy and DNA vaccination. Although it presents enormous advantages in comparison to conventional plasmid DNA regarding bioactivity and safety, its challenging isolation from parental plasmid and miniplasmid has been setting back its launching in biomedical sciences. In this work, it is demonstrated the use of a simple size exclusion chromatographic method for the isolation of supercoiled mcDNA. Sephacryl S-1000 SF matrix was explored under different conditions (flow, peak fractionation volume and sample loading) to achieve the best performance and retrieve a mcDNA sample devoid of other bacterial contaminants or plasmid species resultant from the recombination process. This isolation methodology resulted in 66.7% of mcDNA recovery with 98.1% of purity. In addition, to show the robustness of the method, the potential of using this matrix for the isolation of a larger mcDNA was also evaluated. Upon adjusting the flow or the column volume, the larger mcDNA molecule was also successfully isolated. Overall, a simple and effective strategy has been established for the isolation of supercoiled mcDNA, underlining the potential of size exclusion chromatography in mcDNA separation.

[Primary retroperitoneal tumors. Treatment modality and prognostic factors]. / I tumori retroperitoneali primitivi. Modalità di trattamento e fattori prognostici.

BACKGROUND: Primary Retroperitoneal Tumors (PRT) form a heterogeneous group of malignant mesenchymal and neuroectodermal neoplasms making up only 1% of all solid neoplasms. METHODS: From 1965 to 1997, 27 patients (16 females and 9 males, age range 19-79 years) underwent operations at the General and Cardiovascular Institute of University of Milan for primary retroperitoneal tumors (22 malignant and 5 benign, sarcomas represented 68% of all malignant tumors). A retrospective analysis of these patients was performed to determine the prognostic parameters associated with a favourable prognosis. All patients were admitted with severe abdominal pain or a palpable mass, other symptoms included fewer and weight loss. The median duration of symptoms before presentation was 16 months (range 15 days-5 years). Before surgical treatment abdominal and pulmonary CT scanning and MRN were undertaken on all patients with PRT to define the size of the tumor and the involvement of other retroperitoneal structures. RESULTS: Complete resection was possible in 54% of malignant tumors (n = 12), incomplete resection was performed in 14% (n = 3) and in 31% (n = 7) only biopsy was possible. Recurrent tumor developed in 25% of patients with total resection (n = 3), (median time to recurrence 5 years). Independent treatment variables (size, signs and symptoms, histopathologic findings and grade) and treatment-dependent variables (type of surgical treatment and adjuvant chemotherapy) were analyzed. Patients with complete resection had a 12 month survival of 100% (n = 12) compared to 50% (n = 1) for those undergoing partial resection and 14.2% (n = 1) for those with simple biopsy. A 24-month survival of the patients undergoing complete resection was 25% (n = 3). Median survival for type of surgical treatment was 28 months for complete resection compared to 14 months for partial resection and only 8 months for biopsy. Twelve-months survival for tumor grade was 100% (n = 7) for low grade tumor (G1), compared to 66.6% (n = 4) for median differentiated tumors (G2), and 37.5% (n = 3) for high grade tumors (G3). Twenty-four-months survival was 28.5% (n = 2) for low grade tumors compared to 16.6% (n = 1) of median differentiated tumors. High grade tumors were associated with a 35-month median survival time compared to 17 months for median differentiated tumors and 10 months for low grade tumors. Other variables (histopathologic findings, size, symptoms) had not significant impact on survival. Operative mortality rate was 3.7% (n = 1). The overall 2 years survival rate in patients with totally resected tumors was 25% (n = 3), (2G1, 1G2), while the 5 year survival rate was 16.6% (n = 2), (2G1). CONCLUSIONS: Complete surgical excision and low grade of the tumor are the most important parameters of survival. Recurrent disease is a vexing problem; a careful and prolonged follow-up based on the use of CT and MRN is recommended.

[Prognostic clinical and anatomopathological factors in surgery of carcinoma of the right colon]. / Fattori prognostici clinici ed anatomopatologici nella chirurgia del carcinoma del colon destro.

Between 1965 and 1990, 52 patients were treated for adenocarcinoma of the right colon at the General and Cardiovascular Institute of University of Milan. We have performed in all patients a curative right hemicolectomy. Five year survival rate was 57.7%. Among 12 clinical and pathological variables, the pathologic stage and the degree of cellular differentiation were the most important prognostic factors. Also other factors, like mucin production, tumor ulceration and duration of symptoms were important prognostic factors, because related to a poor prognosis. Our study emphasize the importance of an early diagnosis and a prompt curative surgical treatment to improve patient's survival rate.

[Primary lymphoma of the gastrointestinal tract]. / I linfomi primitivi del canale alimentare.

Lymphomas of the gastrointestinal tract are rare tumors with unusual features and few characteristics. Clinical, radiological and endoscopic diagnosis is difficult, because they may be confused with inflammatory or neoplastic forms, especially in primary involvement of the gastrointestinal tract. From 1965 we observed 4 patients with primitive gastrointestinal tract lymphomas. Prognostically important features for GI lymphomas were: stage, site of the primary disease, histological type. Authors report on the clinical findings, diagnostic procedures, surgical treatment and results.

[Pancreatic insulinomas]. / Gli insulinomi pancreatici.

Neuroendocrine pancreatic tumors are neoplasms derived from APUD cells, characterized by hyperincretion of several peptides of hormonal activity. The incidence of these tumor is low. They are usually classified according to the predominant secreted peptide: gastrinoma, insulinoma, VIPoma, glucagonoma. Insulinoma is the most frequent endocrine pancreatic tumor, characterized by a peculiar clinical picture due to insulin action. This neoplasm is prevalently benign (90%), and may cause symptoms due to hypo-glycemia such as epilepsy, asthenia, deep coma, dizziness, hunger and epigastric pain. Surgery still constitutes the principal therapy for insulinoma treatment, but an accurate tumor identification is necessary. Selective arteriography of the pancreas and new diagnostic investigations as intraoperative US, selective sampling of pancreatic veins with insulin Quick-RIA, aid the diagnosis and more precise localization of the tumor. When surgical therapy is not practicable, for diffuse metastases, octreotide has an inhibitory effect upon hormone release, and may be combined with chemotherapy for controlling clinical symptoms. We review the clinical records of 2 patients from our Institute, who had hyper-insulinism due to benign insulinomas of the tail of the pancreas. Surgical treatment was performed with enucleation of the neoplasms.

[Biliary lithiasis and carcinoma of the gallbladder]. / Litiasi biliare e carcinoma della colecisti.

Gallstones are a major risk factor for gallbladder cancer, but only few patients with stone experience develop this tumor. To examine this relationship, twenty-one patients with gallbladder cancer referred to our Institute over a 26-year period were studied. The tumor was most common in older females (95.2%) (median age 69.2 years). Seventy-six percent of patients had gallstones. The cell type in 57.1% of patients was adenocarcinoma. The prognosis is poor, the median survival time was 6.7 months.

[Acute occlusion of the abdominal aorta]. / L'occlusione acuta dell'aorta addominale.

Acute aortic occlusion is a rare event, usually related to atherosclerotic lesions, cardiac embolism or traumatic events. During the last twelve years, we observed 25 acute aortic occlusions: 12 patients (48%) were affected by aortic bifurcation embolism, and 13 patients (52%) by acute aortic thrombosis. There were 18 men (72%) and 7 women (28%) with a median age of 63 years. Severe cardiopathy was observed in 15 patients (60%). Twenty-four patients (96%) underwent surgical treatment: involvement of the renal arteries was found in only 2 cases (8%). We performed 14 aorto-femoral by-passes (58%). Operative mortality was 20%. Death was due to acute kidney failure in 2 cases and to heart infarction in 3 cases.

[Vascular complications in drug addicts. Intermittent claudication in a HIV seropositive heroin addict]. / Complicanze vascolari in tossicodipendenti. "Claudicatio intermittens" in tossicodipendente da eroina sieropositiva per HIV.

The widespread use of heroin has revealed the growing life-threatening complications consequent on drug practice. The effects of heroin on the cardiovascular, pulmonary, renal and central nervous system have been well described in medical literature, but the vascular complication of chronic occlusive arterial disease of the lower limbs is rare. We report a case of a young female patient who was a chronic drug addict for over five years, with concomitant serum positiveness for HIV infection. The etiologic and pathogenetic mechanisms of such an uncommon vascular complication are discussed on the basis of the existing literature.