A scalable method to concentrate lentiviral vectors pseudotyped with measles virus glycoproteins.

Lentiviral (LV) vectors have emerged as powerful tools for basic research and clinical applications because of their ability to stably transduce both dividing and nondividing cells. A wide range of viral envelope (Env) glycoproteins have the ability to associate with the membrane of LV vectors, a process that is referred to as pseudotyping. Pseudotyped vectors have the capacity to transduce specific cell types for specific applications. For example, LV vectors pseudotyped with the measles virus (MV)-derived hemagglutinin (H) and fusion (F) proteins have the ability to transduce quiescent lymphocytes. In addition, the MV H glycoprotein can be engineered allowing cell-specific targeting of LV vectors. One problem with MV glycoprotein-pseudotyped LV vectors is low titer during vector production. This results in the need to manufacture large volumes of the vectors and to concentrate them to appropriate titers. The commonly used centrifugation-based concentration techniques for LV vectors are not practical for large-scale vector manufacturing. Thus, there is a need for improved methods to concentrate LV vectors. In this study, we adapted an anion-exchange membrane chromatography method that we previously used in the context of LV vectors pseudotyped with the vesicular stomatitis virus glycoprotein to concentate MV glycoprotein-pseudotyped LV vectors. Up to 60% of the input vectors with an up to 5300-fold reduction in volume was achieved using this anion-exchange chromatography method in conjunction with a desalting/concentration step involving centrifugal filter units. This technique provides a rapid and scalable approach for concentrating MV-pseudotyped LV vectors that does not require an elaborate setup.

Rapid titration of retroviral vectors using a ß-lactamase protein fragment complementation assay.

The availability of rapid and quantitative titration assays for retroviral vectors is important, especially in the context of clinical applications. In this report, we describe a novel assay to titrate lentiviral and gamma retroviral vectors. This rapid assay is based on protein fragment complementation involving the N-terminal (Bla1) and the C-terminal (Bla2) fragments of TEM-1 ß-lactamase (BLAK). The Bla1 protein fragment is incorporated in the vector's envelope during vector production. Bla1-bearing vectors are titrated on Bla2-expressing cells. Upon transduction, Bla1 and Bla2 heterodimerize and restore BLAK's enzymatic function. The enzymatic activity of BLAK is quantified by flow cytometry using the green fluorescent CCF2/AM substrate, which is converted into a blue fluorescent product. The enzymatic conversion of the CCF2/AM substrate was found to be directly related to vector entry, as a neutralizing antibody completely blocked the conversion. The titers obtained using this rapid assay correlated well with the titers measured by functional transduction assays. The whole assay can be finished within 8 h. Thus, it is considerably less time consuming compared with other transduction-based titration assays for lentiviral and gamma retroviral vectors.

High prolactin levels as a worsening factor for migraine.

Many factors should be considered when an episodic migraine worsens and becomes chronic. Prolactin (PRL) was linked to the origin of pain in patients with microprolactinomas who developed different types of headaches. Our team carried out studies on 27 patients with a background of episodic headaches that became chronic. The patients were evaluated by means of a general examination, a neurological examination and a hormonal profile. Of the 27 patients, 7 of them had an increased level of prolactinaemia. All the patients were women, ranging from 17 to 57 years of age. Four of them had a pure form of migraine without aura, whereas 3 patients had both migraines without aura and tension-type headaches. They suffered from headache for a period ranging from 3 to 32 years and their headache became chronic 4-12 months prior to the visit. Their headache did not change in type, but only in severity and frequency. Two patients had no symptoms referable to high PRL levels; 4 patients had irregular menses or amenorrhoea. One of these patients also suffered galactorrhoea and two of these patients had a microprolactinoma at MRI; one patient was using estroprogestinic drugs, so her menstrual alteration could not be considered. The patients were followed-up for a period of 6-16 months. Six patients responded favourably after being treated with cabergoline, although some had already tried other drugs, which, however, had no effect on their headache. One patient improved after ceasing to take estroprogestinic, in spite of increased levels of PRL. Therefore, on this basis, PRL levels should always be considered when headache worsens. It is an adjunctive worsening factor, which can be easily eliminated.

Surviving acute myocardial infarction: survivin expression in viable cardiomyocytes after infarction.

BACKGROUND: Apoptosis is a key feature in postinfarction remodelling leading to progressive myocyte loss. Both proapoptotic and antiapoptotic factors contribute to the delicate balance between death and survival. The survivin pathway has emerged as essential in the control of apoptosis, although its role in heart disease is unknown. AIM: To evaluate survivin expression after acute myocardial infarction (AMI). METHODS: Survivin expression was assessed immunohistochemically in the peri-infarct and remote viable myocardium in 17 consecutive patients who died 1-30 weeks after AMI and in four control hearts. RESULTS: Survivin was expressed by myocytes in the peri-infarct area in eight patients and in the remote region in 13 patients. The rate of survivin expression after AMI was significantly higher in the remote versus peri-infarct regions and compared with control hearts. Its expression was inversely associated with the presence of dilated cardiopathy and of apoptosis, independently from the gross pathology infarct size. CONCLUSIONS: Survivin myocardial expression after AMI may be associated with the survival of at risk myocardium and may be indicative of more favourable remodelling after AMI. These findings identify a potential new target for the treatment of postinfarction remodelling.

Right ventricular dilatation after left ventricular acute myocardial infarction is predictive of extremely high peri-infarctual apoptosis at postmortem examination in humans.

BACKGROUND: Cardiac remodelling after acute myocardial infarction (AMI) is characterised by molecular and cellular mechanisms involving both left and right ventricles, and biventricular failure identifies patients with an extremely unfavourable prognosis. AIMS: To assess whether a link exists between increased myocardial apoptotic rates (AR) at sites of recent infarction and patterns of unfavourable cardiac remodelling, such as biventricular enlargement after left ventricular (LV) infarction. METHODS: Twelve patients with recent AMI involving the LV and not the right ventricle (RV) and with permanent infarct related artery occlusion were selected at necropsy. Gross pathological characteristics, such as LV and RV dilatation, and AR at site of infarction were assessed. Potential false positive results (DNA synthesis and RNA splicing) were excluded from the cell count. RESULTS: RV enlargement, defined as a tricuspidal ring greater than 120 mm, was found in five cases and was associated with LV dilatation. These patients showed significantly higher AR than the others. When the subjects were divided into three groups according to progressive cardiac remodelling (absence of cardiac dilatation, isolated LV dilatation, and biventricular enlargement), the last group had significantly higher ARs than the other two groups, showing that myocardiocyte apoptosis is increased in more unfavourable forms of cardiac remodelling. CONCLUSION: Patients with severely unfavourable cardiac remodelling, such as biventricular enlargement, have extremely high myocardiocyte apoptosis at necropsy, even late after LV myocardial infarction, supporting the role of myocardiocyte loss in determining post-infarction adverse remodelling.

Pleomorphic adenoma of cervical heterotopic salivary glands.

Neoplastic transformation of heterotopic cervical salivary gland tissue is extremely rare. The Authors describe a case of pleomorphic adenoma arising in this tissue, on the right side of the neck in a 40-year old man. The characteristics of salivary heterotipias of the neck, and their diagnostic difficulties and histogenetic problems are reviewed.

Expression of p21 in SV40 large T antigen positive human pleural mesothelioma: relationship with survival.

BACKGROUND: Mesothelioma is the most commonly occurring primary pleural neoplasm. Several studies have documented an increase in the incidence of this malignancy during the last decades. Although the association between asbestos exposure and development of mesothelioma is generally accepted, the exact mechanism of carcinogenesis is unknown. Recently, Simian virus 40 large T antigen (SV40 Tag) expression has been detected in pleural mesothelioma. The ability of SV40 oncoproteins to inactivate p53 and retinoblastoma tumour suppressor proteins has been proposed as an important step in the pathogenesis of human mesothelioma. METHODS: To obtain a better understanding of the molecular mechanisms of the pathogenesis of mesothelioma, the expression of the cell cycle inhibitor p21(WAF1/CIP1) (p21), a downstream target of p53, was evaluated immunohistochemically in a group of 29 mesothelioma specimens already characterised for the presence of SV40 Tag sequences. RESULTS: Statistical analysis did not reveal any correlation between p21 expression and histopathological type of mesothelioma using the kappa(2) test (p=0.577). A significant positive relationship was found between p21 expression level and the patients' overall survival according to the Kaplan-Meier survival curves and using a log rank test (median difference in survival 7 months, 95% CI 4.8 to 9.9; p<0.001). CONCLUSIONS: Determination of p21 expression bears a prognostic significance in patients affected with mesothelioma, further underlining the role of SV40 in the pathogenesis of malignant pleural mesothelioma.

Non-alcoholic fatty liver disease in an area of southern Italy: main clinical, histological, and pathophysiological aspects.

BACKGROUND/AIMS: Studies on non-alcoholic fatty liver disease (NAFLD) have included chronic liver damage attributed to various causes. Our investigation was held to observe the main clinical, histological, and pathophysiological aspects of NAFLD in patients not exposed to any known cause of chronic liver disease. METHODS: We evaluated, in 84 in-patients (male/female, 66/18; median age, 36 years), the clinical and biochemical characteristics of NAFLD, and particularly its association with diabetes, dyslipidemia, hyperinsulinemia and/or with the increase of parameters of oxidative stress (blood levels of malonyldialdehyde, 4-hydroxynonenal and total plasma antioxidant capacity). RESULTS: Ninety percent of patients had an increased body mass index (BMI), 35% had dyslipidemia, 40% had sub-clinical diabetes (only 3% had overt diabetes), 60% had hyperinsulinemia, and more than 90% had enhanced levels of lipid peroxidation markers. In 48 patients who had consented to liver biopsy, we found: 14 with simple steatosis, 32 with steatohepatitis, and two with cirrhosis. CONCLUSIONS: Our data indicate that in our country, NAFLD may occur in young males with an increased BMI, with or without hyperinsulinemia, dyslipidemia and diabetes, generally associated with disorders of redox status, and that it may be differentiated from steatosis to steatohepatitis or cirrhosis only with a liver biopsy.

Solitary muscle metastasis from lung carcinoma.

Recurrence after resection of non-small cell lung carcinoma is generally associated with a poor outcome. Limb muscle metastasis from lung cancer is extremely rare. We present a case of a 71-year-old man who presented with a solitary metastasis to his right lower limb two months after right upper lobectomy for lung cancer (stage: T2N0M0). Twenty-four months after surgical excision and chemotherapy he is alive without signs of neoplastic disease. We believe that a more aggressive approach might be considered for selected patients with solitary extracranial and extra-adrenal metastasis from lung cancer.

Drinking habits of subjects with hepatitis C virus-related chronic liver disease: prevalence and effect on clinical, virological and pathological aspects.

Alcohol changes the progression of hepatitis C virus (HCV)-related chronic liver disease and may affect the outcome of interferon therapy. The ethanol intake of 245 patients with biopsy-proven chronic hepatitis C with or without cirrhosis, its interaction with laboratory and histological parameters common to alcohol and HCV-mediated liver damage, and its effects on therapy were evaluated. The results show that 60-70% of subjects regularly consumed alcohol (median intake >40 g/day in about 30%). Less than 50% stopped drinking after being diagnosed as having liver disease. Ethanol intake affected: fibrosis, especially in women, HCV RNA levels, which were significantly lower in abstainers than in drinkers (0.6 +/- 0.3 vs 6.9 +/- 5.9 Eq/ml x10(6); P < 0.01), and response to interferon therapy. The number of responders decreased as ethanol intake increased. There were less abstainers than drinkers among non-responders (10.7% vs 63.1% respectively; P < 0.001). Data indicate that alcohol will induce and worsen liver damage and, in subjects with chronic liver disease who continue to drink, adversely affect their response to treatment.

Atypical fibroxanthoma with osteoclast-like multinucleated giant cells.

Atypical fibroxanthoma (AF) is generally considered as a low grade superficial variant of fibrohistiocytic neoplasm. In this report we present an unusual variant of this neoplasm arising from actinic damaged skin of an elderly individual. The case was characterized by numerous multinucleated osteoclast-like giant cells uniformly scattered through a pleomorfic cellular proliferation. The osteoclasts giant cell observed represent multinucleated histiocytes rather than true osteoclasts.

Neuroendocrine differentiation in non-small cell lung carcinomas.

The immunohistochemical expression of ten neuroendocrine and neural differentiation-related antigens in 200 specimens from patients with surgically resected non-small cell lung carcinomas were evaluated. Poorly differentiated adenocarcinomas and undifferentiated large cell carcinomas showed the highest percentage of positive samples (30%-60%) for the markers evaluated with diffuse and intense immunostaining. Poorly differentiated squamous carcinomas bronchioalveolar adenocarcinomas, and giant cell carcinomas showed a lower percentage of positive samples (20%) with moderate immunostaining. Well differentiated tumors were very rarely positive for the neuroendocrine markers. It is concluded that neoplasms positive for the neuroendocrine markers can be considered as non-small cell carcinoma with neuroendocrine features, even if they do not have the histological appearance of neuroendocrine lung neoplasms.

Relapsing pneumothorax due to Ascaris lumbricoides larvae.

We describe a case of relapsing pneumothorax occurring in a male child aged 11 yrs, resulting from the mechanical inflammatory action of Ascaris lumbricoides larvae localized in the pleuropulmonary tissue. Pulmonary ascariasis should still be considered today in the differential diagnosis of spontaneous pneumothorax occurring in children.

[Basaloid squamous carcinoma of the tongue. Clinico-anatomical study of a case]. / Carcinoma basalioide squamoso della lingua. Studio anatomo clinico di un caso.

The basaloid squamous carcinoma was first described in 1986. It is a rare tumor with particular morphologic and biologic features that separate it from the more common squamous cell carcinoma of the head and neck region. A case with histologic diagnosis of basaloid squamous carcinoma of the tongue treated only with radiotherapy, is reported.

Crohn's disease in infancy: a case report.

Crohn's disease in infants is infrequent. We describe here the case of a baby girl born at the 33rd week of pregnancy, that underwent surgery at the age of 55 days for intestinal occlusion. This was found to be due to stenosis of the right colonic flexure associated with cecum, appendix and terminal ileum. Microscopic examination showed features consistent with Crohn's disease. Eleven years after surgery, followed by cortisone therapy, the patient shows normal growth and is asymptomatic.

Lymphomatoid granulomatosis of the lung: a clinico-pathological study.

Lymphomatoid granulomatosis is the only form of pulmonary angiitis histologically characterized by a necrotizing angiocentric and angiodestructive lymphoid infiltrate, with an admixed T-cell reaction. We evaluated three patients with a single lung nodule not diagnosed by routine radiological and endoscopic assays. Our investigations showed a prevalence of T-cells in areas of diffuse infiltration, which were actively replacing reactive follicular areas of B-cells, similarly to T-cell lymphomas. Further pathologic assays suggested the histologic diagnosis of grade I lymphomatoid granulomatosis for all three evaluated specimens. After two years, patients treated with a combination of surgical resection and chemotherapy were disease free, supporting the efficacy of aggressive therapy in the management of this often mistreated group of lymphoid proliferations.

[Pulmonary blastoma in adults]. / Blastoma polmonare dell'adulto.

Pulmonary blastoma is a rare tumor of the lung consisting of immature mesenchyma and epithelial structures that can mimic the structure of the embryonic lung. In this article, we report a case of pulmonary blastoma occurred in an adult individual. We review the literature concerning this rare neoplasm that includes less than one hundred cases reported. We also describe the pathologic features of this disease which is microscopically divided in two classes: 1) lesions composed solely of malignant gland of embryonic appearance (WDFA: Well-Differentiated Fetal Adenocarcinomas), and 2) lesions with biphasic appearance, containing both neoplastic glands and either adult sarcomatous or embryonic mesenchyme. The adult pulmonary blastomas are considered as an entity distinct from childhood blastomas. This condition may be frequently asymptomatic; if symptomatic, it show the same clinical picture of a mass in the lung impinging on bronchi or pleura; the most usual symptoms include cough, hemoptysis, and chest pain. Chest radiography typically shows a peripheral or central lung opacity without preference for any lobe. The prognosis is poor, due to the high incidence of metastasis. Histologic class and clinical findings can be of prognostic value in pulmonary blastoma. We believe that an early diagnosis is essential: in our case, this allowed a complete resection of the tumor and the patient is still alive without recurrence 3 years after the surgical treatment.

[Cystic adenomatoid malformation of the lung in an adult]. / Malformazione adenomatoide cistica del polmone in un adulto.

Congenital cystic adenomatoid malformation of the lung (CCAM) is characterized by an adenomatoid proliferation of bronchiole-like structures and cysts formation. The condition is most commonly found in newborns and children and it may be associated with other malformations; rarely, the presentation is delayed until adulthood. This paper presents a case of CCAM in a 62-year-old male, who presented with recurrent bacterial pneumonias and breathlessness one exertion. The chest X-rays and CT scan revealed a patchy opacity in the right lower pulmonary zone. Bronchoscopic examination was normal. At surgery, a mass involving the right lower and middle lobes, and enlargement of hilar lymph nodes were found. A bilobectomy was performed without complications. Examination of the gross specimen showed a lesion characterised by multiple small cysts, all less than 1 cm in diameter; they were lined predominately by columnar epithelium, occasionally by ciliated epithelium. Rare cysts were lined by foreign body giant cells. Elastic fibers and smooth muscle were present within the cysts wall. Peripherally, there were normal alveoli and bronchioli mixed with cysts, and plasmalymphocytic infiltrates. The final diagnosis was Stocker's Type II CCAM of the lung. CCAM of the lung is a rare development lesion of the lung and it has no sexual predilection. It is usually unilateral and sublobar or lobar in size, but occasionally it can be multilobar. Typical histologic feature of CCAM are adenomatoid proliferation of bronchiole-like structures and macro- or microcysts lined by columnar or cuboidal epithelium and absence of cartilage and bronchial glands. Inflammatory changes are not found in infants, but may be present in adult patients. Based on the size of the cysts, CCAM may be classified into three different types: type I characterised by multiple cysts, over than 1 cm in diameter; type II with smaller cysts, less than 1 cm in diameter; type III that shows solid lesions composed of bronchiole-like structures. Type II is commonly found in childhood, but is occasionally seen in adult patients, as that one in our report. The insult probably occurs between 4th and 7th week of fetal life. The etiologic agent is unknown. The histologic diagnosis of CCAM is difficult in adult patient, perhaps because of supervening infections that sometimes distort the underlying diagnostic pathologic appearances and make them difficult to recognise, as happened in our case. From the clinical point of view, most of the lesions cause severe respiratory failure; in adult individuals the diagnosis is difficult, since there are very few relevant symptoms and signs. The patients can present with fever, recurrent infections, breathlessness and haemoptysis. The chest X-rays abnormalities are not specific and include homogeneous or multicystic opacities. Similarly, other diagnostic methods add no further useful informations. Surgical treatment is necessary also in adult patients, because of the risk of recurrent pulmonary infections and malignancies associated with CCAM. Lobectomy is the treatment of choice, but sometimes a larger resection is required, when the lesion involves more than one lobe.

Subchorionic placental cyst: histopathological and clinical aspects in two cases.

Authors report here two cases of subchorionic placental cysts with diameters of 5 x 6 cm and 10 x 12 cm, respectively, diagnosed by ultrasound in pregnancy. Subsequent histological and biochemical examinations seem to prove their origin from the trophoblastic "X-cells" and confirm their frequent benign nature.