Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression

The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3′ end of the AAV genome. This protein, designated the single-stranded D-sequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.     

Challenges in the Local Treatment of Large Abdominal Embryonal Rhabdomyosarcoma

Background

Embryonal rhabdomyosarcoma is the most common pediatric soft tissue sarcoma. The best local treatment in large, nonmetastatic primary unresected nongenitourinary embryonal rhabdomyosarcoma of the abdomen (LARME) is however unclear.

Methods

We analyzed patients with LARME treated in four consecutive CWS trials. All diagnoses were confirmed by reference reviews. Treatment included multiagent chemotherapy and local treatment of the primary tumor with surgery and/or radiotherapy. The impact of primary debulking surgery (PDS) also was studied.

Results

One hundred patients <21 years with a median age of 4 years had LARME. Sixty-one of them had a tumor >10 cm in diameter at diagnosis. PDS was performed in 19 of 100 children. The outcomes of patients with PDS were similar to those of the other patients. In 36 children, the tumor was resected after induction chemotherapy; 60 RME were irradiated. The toxic effects of radiochemotherapy were not significantly increased compared with the nonirradiated patients. With a median follow-up of 10 years, the 5-year EFS and OS were 52 ± 10 and 65 ± 9 %, respectively. Significant risk factors in multivariate analysis were age >10 years; no achievement of complete remission; and inadequate secondary local treatment, defined as incomplete secondary resection or no radiation.

Conclusions

Children with LARME have a fair prognosis, despite an often huge tumor size and unfavorable primary site, if the tumors can either be resected or irradiated following induction chemotherapy. PDS was only performed in a small subgroup. Radiation performed concomitantly with chemotherapy did not increase the acute toxicity significantly.     

Prognostic Factors Assessed for 15,096 Patients with Colon Cancer in Stages I and II

Background

We focused on the risk factors for poor outcome after curative resection of a colon cancer in UICC stages I and II based on the data of the Germany-wide quality assurance study “colon/rectum cancer (primary tumor).” In some countries, all stage II colon cancer patients are encouraged to participate in a clinical trial. We feel that this approach is too broad.

Methods

Using the data of 15,096 patients operated on from January 1, 2000 to December 31, 2004, the following factors were analyzed with the Cox regression model: age, comorbidities, ASA score, gender, localization of the tumor (left colon vs. right colon), perioperative complications (yes/no), pT stage, grading (G1/G2 vs. G3/G4), L-status (lymph vessels invasion yes/no), and V-status (venous invasion yes/no).

Results

The probability of a local relapse in stages I and II was 1.5 and 4.6%, respectively, or distant metastases 4.7 and 10.2%, respectively. Only pT stage [hazard ratio (HR) for pT1?=?1, pT2?=?1.821, pT3?=?2.735, and pT4?=?5.881], L-status (HR for L1?=?1.393), age (HR per year?=?1.021), as well as ASA score IV (HR?=?4.536) had significant influence on tumor-free survival.

Conclusions

Despite favorable prognosis and R0 resection, a small percentage of patients will still relapse. The most important risk factor comprising the tumor-free survival is the pT stage followed by L-status and age. These results should be taken into consideration when determining the course for adjuvant chemotherapy, especially if the course includes the recommendation of clinical trial participation for stage II colon cancer patients after an R0 resection.     

Laparoskopische Stomaanlage – eine ideale Einzelindikation in der minimal-invasiven Chirurgie

Die laparoskopische Anlage eines Ileostomas bzw. Colostomas stellt ein schnelles, sicheres und technisch einfaches Verfahren dar. Die Vorteile der minimal-invasiven Vorgehensweise liegen in einer niedrigen Morbidit?t und Mortalit?t, einem minimalen Blutverlust, einer schnellen Rekonvaleszenz dieser h?ufig multimorbiden Patienten und einer verkürzten Hospitalisierungsdauer. Wir berichten über eine Serie von 20 Patienten innerhalb eines 2-Jahres-Zeitraums, bei denen in 19 F?llen ein Colostoma und in 1 Fall ein Ileostoma angelegt wurden. Es traten keine intraabdominellen Komplikationen auf. Eine Hospitalletalit?t gab es nicht. Alle Patienten erreichten sp?testens am 2. postoperativen Tag ihren pr?operativen Mobilisierungsgrad. Die Indikationen für eine laparoskopische Enterostomie werden dargestellt.     

Attention deficit hyperactivity disorder: comorbidity and medication use

Children evaluated for attention deficit hyperactivity disorder (ADHD) may have other disorders resembling ADHD leading to inappropriate stimulant medication use. This study was completed to identify relationships between referral complaints of ADHD, behavior problems or learning problems and age, gender, final diagnosis, and medication use. One hundred eighty-nine children ages 2 to 15 years referred for evaluation of ADHD, behavior or learning problems were evaluated by an interdisciplinary team. Diagnoses of ADHD, specific learning disability (SLD), mental retardation (MR), developmental language disorders (LANG), and other behavior disorders (DIS) were established. Medication use pre- and post-evaluation was reviewed. Forty-three percent of all subjects had a final diagnosis of ADHD. Forty percent referred specifically for presumed ADHD did not have it. More children older than 5 years were diagnosed as having ADHD than those 5 years old or younger (p < 0.0001). More subjects 5 years old or younger were diagnosed as having LANG than those older than 5 years (p < 0.0001). Fewer subjects with a chief complaint of ADHD were diagnosed with MR than those with behavior or learning problems (p = 0.001). In subjects 5 years old or younger, 35% were diagnosed with MR and 49% with other DIS. In children older than 5 years, 41% were diagnosed with SLD. Ten percent of subjects without ADHD were using stimulants. Only 48% of subjects with confirmed ADHD took stimulants. Children presenting with behavior problems or those 5 years old or younger are at higher risk for MR, LANG, and DIS and less likely to have ADHD. Children presenting with learning problems or those older than 5 years are more likely to have SLD or ADHD. Multiple diagnoses were common for all ages and presentations. Ten percent of children without confirmed ADHD used stimulants before evaluation.     

Retrieval of migrated ureteral stent by intussusception of ureteral balloon dilator tip

A vexing complication of ureteral stent placement is inadvertent proximal migration. The retrieval of a migrated ureteral stent may be performed using a myriad of techniques but usually requires ureteroscopic extraction. We report a novel, time-efficient technique to capture and reposition a migrated ureteral stent without ureteroscopic manipulation.     

Continuous Intrathecal Baclofen Treatment of Severe Spasms in Two Children with Spinal-cord Injury

This study reports the use of intrathecal baclofen in two ventilator-dependent children with severe spasms secondary to spinal-cord injury. Baclofen was delivered via a subcutaneously implanted, programmable pump. The children were followed for 12 and 24 months. Baclofen dramatically reduced spasms, resulting in more stable ventilation, improved ease of care, reduced distress and better integration into the community. Although effective, intrathecal baclofen represents a significant intervention; careful consideration must be given to potential complications and the need for long-term management. Full effectiveness was dependent on free CSF flow.     

Simultaneous analysis of interleukin-10 gene microsatellites and single-nucleotide polymorphisms in parallel with tumour necrosis factor and interferon-gamma short tandem repeats by fluorescence-based polymerase chain reaction

Different cytokine genotypes exist in the population, for example, as a result of selective pressure of infectious diseases. It may be that specific cytokine genotypes that are beneficial by creating a 'proinflammatory' phenotype predispose to severe inflammatory disease with worse clinical outcome. There is individual variation in the production of certain cytokines in relation to their genotypes. IL-10, IFN-gamma and TNF-alpha are key components in the regulation of immune responses and the balance of their expression levels is predictive in certain diseases. To describe cytokine genotypes, a one-tube PCR reaction was developed to analyse simultaneously DNA sequence variations of cytokine genes IL-10, IFN-gamma, and TNF. This multiplex PCR approach was used to provide genotypic data for two geographically independent donor groups from Germany and Gabon. Significant differences were obtained for the majority of sequence variations comparing both populations. However, the SNPs within the 5'-flanking region of the IL-10 gene at position -1087 and -6208 are comparable in their genic and genotypic behaviour. Comparing allelic and genotypic disequilibrium between pairs of loci revealed different association patterns for both populations according to the geographical polymorphism. This assay may improve immunogenetic studies in disease, characterized by disbalanced IL-10, IFN-gamma and TNF-alpha expression.     

Capnography during cardiac resuscitation: a clue on mechanisms and a guide to interventions

Measurement of the end-tidal partial pressure of carbon dioxide (P_ETCO₂) during cardiac arrest has been shown to reflect the blood flow being generated by external means and to prognosticate outcome. In the present issue of Critical Care, Grmec and colleagues compared the initial and subsequent P_ETCO₂ in patients who had cardiac arrest precipitated by either asphyxia or ventricular arrhythmia. A much higher P_ETCO₂ was found immediately after intubation in instances of asphyxial arrest. Yet, after 1 min of closed-chest resuscitation, both groups had essentially the same P_ETCO₂, with higher levels in patients who eventually regained spontaneous circulation. The Grmec and colleagues' study serves to remind us that capnography can be used during cardiac resuscitation to assess the mechanism of arrest and to help optimize the forward blood flow generated by external means.