Intracranial dural based marginal zone MALT-type B-cell lymphoma: a case - Based update and literature review.

OBJECTIVES: Dural based Marginal Zone MALT-type B-Cell Lymphoma (MZBCL) is an intracranial tumor that can mimicking meningioma both from a clinical and a radiological point of view. A standard treatment protocol is still lacking. Aim of the present work is to provide an update of the present literature regarding this rare neoplasia. PATIENTS AND METHODS: We report the case of a patient with a dural-based lesion mimicking a meningioma of the tentorium. After surgical treatment, the diagnosis was of MZBCL. A literature review is performed to highlight the typical characteristics of this rare intracranial lesion and to define the best therapeutic approach. RESULTS: Literature review included 38 articles describing 126 cases of intracranial dural-based MZBCL. No clinical trial has been found. Clinical and histopathological features are properly collected to provide a guide for future cases. Different treatment options have been attempted. Combination of surgery with adjuvant radiation therapy is the most used option. CONCLUSIONS: MZBCL should be considered in differential diagnosis for dural-based intracranial lesion. Surgery followed by radiation therapy is the most reported treatment. As a consequence of the rarity of this disease, of its indolent progression and of the lack of adequate follow-up, it is not possible to define it is the best treatment option.

Trans-sulcal versus trans-parenchymal approach in supratentorial cavernomas. A multicentric experience.

OBJECTIVES: Cavernous malformations (CM) are low-flow vascular lesions that can cause significant symptoms and neurological deficits. Different intraoperative surgical approaches have been developed. Aim of the present investigation is the comparison between the trans-sulcal approach (TS) and the trans-parenchymal neuronavigation-assisted approach (TPN) in a surgical series from two neurosurgical centers. The technique and clinical outcomes are discussed, with a specific focus on seizure outcome. PATIENTS AND METHODS: Clinical and radiological data from two neurosurgical centers ("A. Gemelli" Hospital in Rome and A.O.U. Città della Salute e della Scienza in Turin) were retrospectively reviewed in order to evaluate the different outcome of TS and TPN approach for cavernous malformation treatment. RESULTS: A total of 177 patients underwent surgical intervention for supratentorial CM, 130 patients with TPN approach and 47 with TS approach. TS approach was associated with higher rate of seizure in early post-operative period both in epileptic patients (p < 0,001) and in patients without history of seizures before surgery (p = 0,002). Moreover, length of incision (p < 0,001), area of craniotomy (p < 0,001) and corticectomy (p < 0,001) were bigger in TS than in TPN approach. Brain contusion (p < 0,001) and fluid collection (p < 0,001) were more likely to be discovered after TS approach. CONCLUSIONS: TPN is a valuable approach for resection of CM. Minor complications are significantly lower in TPN approach when compared with TS approach. In addition, it is associated with lower rate of early post-operative seizure and shorter length of stay.

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.

Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors.

Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.

Novel adeno-associated viral vectors for retinal gene therapy.

Vectors derived from adeno-associated virus (AAV) are currently the most promising vehicles for therapeutic gene delivery to the retina. Recently, subretinal administration of AAV2 has been demonstrated to be safe and effective in patients with a rare form of inherited childhood blindness, suggesting that AAV-mediated retinal gene therapy may be successfully extended to other blinding conditions. This is further supported by the great versatility of AAV as a vector platform as there are a large number of AAV variants and many of these have unique transduction characteristics useful for targeting different cell types in the retina including glia, epithelium and many types of neurons. Naturally occurring, rationally designed or in vitro evolved AAV vectors are currently being utilized to transduce several different cell types in the retina and to treat a variety of animal models of retinal disease. The continuous and creative development of AAV vectors provides opportunities to overcome existing challenges in retinal gene therapy such as efficient transfer of genes exceeding AAV's cargo capacity, or the targeting of specific cells within the retina or transduction of photoreceptors following routinely used intravitreal injections. Such developments should ultimately advance the treatment of a wide range of blinding retinal conditions.

AAV-mediated photoreceptor transduction of the pig cone-enriched retina.

Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy of retinal diseases caused by defects in the retinal pigment epithelium (RPE). In contrast, evidence of the efficacy of AAV-mediated gene transfer to retinal photoreceptors, the major site of inherited retinal diseases, is less robust. In addition, although AAV-mediated RPE transduction appears efficient, independently of the serotype used and species treated, AAV-mediated photoreceptor gene transfer has not been systematically investigated thus so far in large animal models, which also may allow identifying relevant species-specific differences in AAV-mediated retinal transduction. In the present study, we used the porcine retina, which has a high cone/rod ratio. This feature allows to properly evaluate both cone and rod photoreceptors transduction and compare the transduction characteristics of AAV2/5 and 2/8, the two most efficient AAV vector serotypes for photoreceptor targeting. Here we show that AAV2/5 and 2/8 transduces both RPE and photoreceptors. AAV2/8 infects and transduces photoreceptor more efficiently than AAV2/5, similarly to what we have observed in the murine retina. The use of the photoreceptor-specific rhodopsin promoter restricts transgene expression to porcine rods and cones, and results in photoreceptor transduction levels similar to those obtained with the ubiquitous promoters tested. Finally, immunological, toxicological and biodistribution studies support the safety of AAV subretinal administration to the large porcine retina. The data presented here on AAV-mediated transduction of the cone-enriched porcine retina may affect the development of gene-based therapies for rare and common severe photoreceptor diseases.

Estimates of lifetime attributable risk of cancer after a single radiation exposure from 64-slice computed tomographic coronary angiography.

AIMS: To estimate the life attributable risk (LAR) of cancer incidence over a wide range of dose radiation exposure and a large spectrum of possible diagnostic computed tomographic coronary angiography (CTCA) scenarios. METHODS: This study included 561 consecutive patients who underwent a successful prospective ECG-gating CTCA protocol (low-dose group) 64-slice CTCA and 188 patients who underwent retrospective ECG-gating CTCA with ECG-triggered dose modulation CTCA (high-dose group). LAR was computed, given the organ equivalent dose, for all cancers in both sexes. LAR was tabulated for each decile of dose-length product by 10-year age classes, separately for each sex. RESULTS: Estimates of LAR of any cancer for an exposure at age < or =40 year were lower in males than in females for any given quantile. At age >/ or =50 years, LAR was similar between sexes only at the lowest exposure doses, whereas at higher dosage, it was, in general, higher for women. At the median age of this case series (62 years) and for a radiation exposure ranging from 1.33 to 3.81 mSv, LAR was 1 in 4329 (or 23.1 per 10(5) persons exposed) and 1 in 4629 (or 21.6 per 10(5) persons) in men and women, respectively. For an exposure ranging from 10.34 to 18.97 mSv at the same median age, the LAR of cancer incidence was 1 in 1336 (or 74.8 per 10(5) persons) in men and doubled (1 in 614 or 162.8 per 10(5) persons) in women. CONCLUSIONS: This study provided an estimate of the LAR of cancer in middle-aged patients of both sexes after a single diagnostic CTCA, providing an easy-to-read table.

Isolation and evaluation of novel adeno-associated virus sequences from porcine tissues.

High antigenic compatibility and low toxicity is associated with xenograft transplantation of porcine tissues in immunodeficient human recipients. We hypothesized that adeno-associated viruses (AAVs) of porcine origin could be highly compatible to human tissues and thus of good efficiency and low toxicity for in vivo gene transfer. Porcine tissues were screened by PCR for the presence of AAV using primers designed to bind conserved regions and amplify variable regions of an alignment of several AAV sequences available on GenBank. We isolated new AAV capsid sequences from porcine tissues and successfully generated a recombinant AAV2/po1 vector by transfection. The AAV2/po1 vector was not cross-neutralized by antisera generated against all other commonly used AAVs (serotype 1, 2, 3, 4, 5, 7 and 8) indicating a distinct antigenic profile. Preexisting immunity to AAVpo1 could not be detected in the human sera evaluated. In mice, AAV2/po1 particles expressing beta-galactosidase or green fluorescent protein demonstrated high transduction efficiency in muscle fibers and the retina after intramuscular or intraocular administration. Biodistribution experiments following systemic administration showed efficient gene transfer exclusively in muscle fibers. Novel AAVs derived from porcine tissues may contribute to the generation of new preventive or curative clinical modalities acceptable for human use.

Replacement of a severe chronic post-traumatic aneurysm of the ascending aorta with aortic valve conduit--reconstruction of the anterior mitral valve ring and implantation of A-V sequential/biventricular pacemaker.

We present the case of a 23-year-old African professional footballer who was admitted on April 1, 1999 to the Cardiology Department of the University Hospital in Magdeburg, on an emergency basis, from a regional lung clinic. According to the history, he was involved in a collision with an opposing player during a football match in his country (in Africa). He lost consciousness for a short time, but continued playing to the end of the match. About two months later he was invited by a German football club for a check-up, with the view to ultimately playing for the club. The team did not find him physically fit enough to play professional football, so he decided to go to Paris by bus on March 31, 1999. During the journey he suddenly became cardio-pulmonary decompensated and had to undergo cardio-pulmonary resuscitation (CPR). He was intubated and placed on a respirator and immediately transferred to a nearby lung clinic. From the lung clinic he was transferred to the Intensive Care Unit of the Cardiology Department of the Magdeburg University Hospital, on April 1, 1999 as an emergency case. He was intensively treated with catecholamines, intravenous ACE inhibitors and diuretics. His clinical condition did not improve appreciably. His chest X-ray showed extreme dilatation of the right and left heart as well as extreme pulmonary congestion.

Efficient trans-splicing in the retina expands the utility of adeno-associated virus as a vector for gene therapy.

Recombinant vectors based on adeno-associated virus (AAV) can efficiently transduce many different cell types, including cells of the retina, resulting in stable gene expression. A major shortcoming of this vector is its small packaging capacity. A trans-splicing approach, which reconstitutes gene expression from two independent AAV vectors, can be used to overcome the vector's packaging limitations. The efficiency of this system to date has been disappointing, and therefore its utility for therapeutic application limited. We demonstrate here that efficiency and cellular specificity of trans-splicing is dependent on selection of the appropriate AAV serotype. Efficiency of transgene expression resulting from trans-splicing in skeletal muscle approaches that obtained when delivering the intact transgene when using AAV2 vectors packaged with AAV5 capsids (AAV2/5). This expands the potential of AAV vectors for retinal gene therapy. The use of AAV2/5 also increases the efficiency of trans-splicing in photoreceptors. Selection of the appropriate AAV serotype is likely to affect efficiency of trans-splicing in other organ systems as well.

Constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer.

To test whether hepatocytes engineered in vivo can serve as surrogate beta cells by similarly secreting mature insulin in a glucose-sensitive manner, we prepared adenoviral vectors encoding wild-type proinsulin (hIns-wt), a modified proinsulin cleavable by the ubiquitously expressed protease furin (hIns-M3), or each of the two beta cell specific pro-insulin convertases PC2 and PC3. Following a detailed in vitro characterization of the proteins produced by our vectors, we infected the liver and, for comparison, the muscle of a chemically induced murine model of type I diabetes. Insulin expression from the transduced tissues was extensively characterized and showed to be constitutive rather than regulated. To obtain regulated expression, we placed expression of hIns-M3 under the control of the dimerizer-inducible transcription system. Hormone secretion from mouse liver was negligible in the absence of the dimerizer drug rapamycin, was inducible in a dose-dependent manner upon its administration, and reversible following drug withdrawal. These data confirm liver as a promising target for in vivo expression of processed insulin. While suggesting that hepatocytes cannot provide authentic glucose-responsive regulation, these results demonstrate that pharmacological regulation is a promising alternative route to the controlled delivery of insulin following hepatic gene transfer.

Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model.

Recombinant vectors based on adeno-associated virus (AAV) or human immunodeficiency 1 (lentivirus) are promising tools for long term in vivo gene delivery. Their design allows the exchange of capsids or envelopes, respectively, theoretically providing the opportunity to transduce a range of cell types. We constructed AAV vectors encoding enhanced green fluorescent protein (EGFP) within an AAV serotype 2 (AAV2) genome contained in an AAV2, five or one capsid (called AAV2/2, AAV2/5 and AAV2/1, respectively). Similarly we produced lentiviral vectors, encoding the same expression cassette present in the AAV vectors, pseudotyped with proteins from vesicular stomatitis virus glycoprotein (VSVG) or Mokola envelopes. Transduction characteristics of these vectors were evaluated in the murine retina following subretinal or intravitreal administration. The time of onset of transgene expression and the targeted cell types differed between the various recombinants. Onset of transgene expression was 3-4 days for lentiviral vectors and AAV2/1. In contrast, onset was at 2-4 weeks for AAV2/5 and AAV2/2, respectively. After subretinal injection, both lenti-VSVG and AAV2/5 transduced the retinal pigment epithelium (RPE) and photoreceptors efficiently whereas transgene expression was restricted to RPE cells using lenti with the Mokola envelope or AAV2/1. After intravitreal administration, only AAV2/2 and lenti-VSVG transduced the inner retina. Vector-mediated fluorescence was detected in the retina for over 12 weeks for all of the vectors. We conclude that pseudotyping provides a useful means to manipulate viral vector cell targeting specificity as well as retinal transduction characteristics of vectors containing the same genome.

A single-step affinity column for purification of serotype-5 based adeno-associated viral vectors.

Here we describe a single-step affinity column for purification of vectors based on adeno-associated virus type 5 (AAV5). A sialic-acid-rich protein called mucin was covalently attached to Sepharose and was found to bind AAV5 vectors. Elution with high salt efficiently recovered highly active vectors of greater purity than what is achieved with CsCl(2) sedimentation.

Adenoviral vector-mediated insulin gene transfer in the mouse pancreas corrects streptozotocin-induced hyperglycemia.

Therapy for type 1 diabetes consists of tight blood glucose (BG) control to minimize complications. Current treatment relies on multiple insulin injections or an insulin pump placement, beta-cell or whole pancreas transplantation. All approaches have significant limitations and have led to the realization that novel treatment strategies are needed. Pancreatic acinar cells have features that make them a good target for insulin gene transfer. They are not subject to autoimmune attack, a problem with pancreas or islets transplantation, they are avidly transduced by recombinant adenoviral vectors, and capable of exporting a variety of peptides into the portal circulation. Recombinant adenoviral vectors were engineered to express either wild-type or furin-modified human insulin cDNA (AdCMVhInsM). Immunodeficient mice were made diabetic with streptozotocin and injected intrapancreatically with the vectors. BG and blood insulin levels have normalized after administration of AdCMVhInsM. Immunohistochemistry and electron microscopy showed the presence of insulin in acinar cells throughout the pancreas and localization of insulin molecules to acinar cell vesicles. The data clearly establish a relationship between intrapancreatic vector administration, decreased BG and elevated blood insulin levels. The findings support the use of pancreatic acinar cells to express and secrete insulin into the blood stream.

Hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer.

Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.

Pharmacologically regulated gene expression in the retina following transduction with viral vectors.

The availability of inducible expression systems makes regulatable control of therapeutic proteins an attainable goal in gene therapy. We delivered tetracycline-inducible transgenes to the subretinal space using recombinant adenoviruses. Upon administration of doxycycline, we demonstrated reversible expression of green fluorescent protein in the retinal pigment epithelium as well as modulation of human growth hormone produced in the retina and secreted in the blood stream. This mode of delivery and regulation offers a unique way to evaluate gene function in the eye and represents a novel method for introducing therapeutic proteins into the retina.

In vivo detection of gene expression in liver by 31P nuclear magnetic resonance spectroscopy employing creatine kinase as a marker gene.

In vivo assessment of gene expression is desirable to obtain information on the extent and duration of transduction of tissue after gene delivery. We have developed an in vivo, potentially noninvasive, method for detecting virally mediated gene transfer to the liver. The method employs an adenoviral vector carrying the gene for the brain isozyme of murine creatine kinase (CK-B), an ATP-buffering enzyme expressed mainly in muscle and brain but absent from liver, kidney, and pancreas. Gene expression was monitored by (31)P magnetic resonance spectroscopy (MRS) using the product of the CK enzymatic reaction, phosphocreatine, as an indicator of transfection. The vector was administered into nude mice by tail vein injection, and exogenous creatine was administered in the drinking water and by i.p. injection of 2% creatine solution before (31)P MRS examination, which was performed on surgically exposed livers. A phosphocreatine resonance was detected in livers of mice injected with the vector and was absent from livers of control animals. CK expression was confirmed in the injected animals by Western blot analysis, enzymatic assays, and immunofluorescence measurements. We conclude that the syngeneic enzyme CK can be used as a marker gene for in vivo monitoring of gene expression after virally mediated gene transfer to the liver.

Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column.

One of the most promising gene transfer vectors in human clinical trials is AAV2. The quality of the vector preparations is a key element in obtaining reliable and reproducible data in preclinical studies. However, established protocols either result in impure, low infectious virus (CsCl2 gradient centrifugation) or demand a high level of manual and technical skills (CsCl2 gradient centrifugation, iodixanol/heparin or HPLC purification). In this study, we present an easy-to-do single-step column purification (SSCP) of AAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation. Various vector preparations generated by our method reproducibly showed high titers, infectivity, and purity. In vivo, our single-step column-purified AAV2 vectors mediate significantly higher transduction efficiency compared with conventional protocols. Investigators still unsatisfied with previously published techniques or new to the field of AAV production may find in our method an interesting alternative.

Circadian variations of ventricular tachyarrhythmias detected by the implantable cardioverter-defibrillator.

OBJECTIVE: The purpose of this study was to prospectively evaluate the daily distribution of episodes of ventricular tachyarrhythmias triggering a last-generation ICD implanted in survivors of cardiac arrest not receiving any antiarrhythmic drug. BACKGROUND: Previous studies reported a circadian variation of out-of-hospital sudden cardiac arrest. Survivors of cardiac arrest and patients with sustained ventricular tachyarrhythmias have a higher risk of recurrence, but there is a lack of detailed information on the daily distribution of ventricular arrhythmia capable of precipitating the recurrence of a malignant ventricular event. METHODS: We used the data stored by a last-generation implantable cardioverter-defibrillator (ICD) to prospectively evaluate circadian distribution of ventricular tachyarrhythmias in 30 survivors of cardiac arrest, implanted with an ICD during an antiarrhythmic drug-free period. RESULTS: During a follow-up of 475 +/- 127 days, a total of 571 ventricular arrhythmias was recorded. Of these stored events, 39 episodes were ventricular fibrillation (VF), 428 events were ventricular tachycardia (VT) and the remaining 104 device activations were VTs spontaneously terminating before device therapy. A circadian variation (p < 0.001) of episodes of ventricular tachyarrhythmias was evident. The incidence of ventricular arrhythmias sharply increased at 6 a.m. and reached a maximum 4 hours later, after which there was a short decline and then a small peak between 3 p.m. and 5 p.m. With respect to different arrhythmias, VF or VTs with a cycle length < or = 350 msec demonstrated a circadian variation. Instead, VTs with a cycle length longer than 350 msec or spontaneously terminating before device discharge had a more even distribution throughout the day. In addition, most of the VF or VTs with a cycle length < or = 350 msec occurred within a few hours after awakening, which was not the case for VTs with a cycle length longer than 350 msec. CONCLUSIONS: The data of this study clearly show the existence of different circadian variations in the occurrence of ventricular tachyarrhythmias, suggesting complex interactions between the autonomic nervous system, ventricular electrophysiological properties and the onset of arrhythmia.