Adeno-associated virus vector-mediated systemic delivery of IFN-beta combined with low-dose cyclophosphamide affects tumor regression in murine neuroblastoma models.

PURPOSE: Type I IFNs (IFN-alpha/beta) have shown significant antitumor activity in preclinical models but limited efficacy and significant toxicity in clinical trials. We hypothesized that the antitumor activity of type I IFNs could be enhanced by chronic, low-dose systemic delivery and sought to test this in murine neuroblastoma models. EXPERIMENTAL DESIGN: Continuous liver-generated expression of human IFN-beta (hINF-beta) was achieved through a gene therapy-mediated approach using adeno-associated virus vectors encoding hIFN-beta (AAV hINF-beta). Orthotopic localized retroperitoneal and disseminated models of neuroblastoma were established using three different xenografts. Immunohistochemical analysis and ELISA were used to evaluate the antiangiogenic effect of therapy. RESULTS: The development of both localized orthotopic (retroperitoneal) and disseminated neuroblastoma was prevented in all mice expressing hINF-beta. Continued growth of established retroperitoneal tumors, treated with AAV hINF-beta as monotherapy, was significantly restricted, and survival for mice with established, disseminated disease was significantly prolonged following administration of AAV hINF-beta. Analysis of treated tumors revealed a significant antiangiogenic effect. Mean intratumoral vessel density was diminished and expression of the angiogenic factors vascular endothelial growth factor and basic fibroblast growth factor were both decreased. Finally, combination therapy in which AAV hIFN-beta was used together with low-dose cyclophosphamide resulted in regression of both established retroperitoneal and disseminated disease. CONCLUSIONS: AAV-mediated delivery of hIFN-beta when used as monotherapy was able to restrict neuroblastoma growth due in part to inhibition of angiogenesis. When used in combination with conventional chemotherapy, AAV hIFN-beta was able to effect complete tumor regression.     

Positron emission tomographic investigations of central muscarinic cholinergic receptors with three isomers of [76Br]BrQNP

We studied the potential of three radiobrominated isomers of BrQNP, (Z(-,-)-[⁷⁶Br]BrQNP,E(-,-)-[⁷⁶Br]BrQNP andE(-,+)-[⁷⁶Br]BrQNP), as suitable radioligands for imaging of central muscarinic cholinergic receptors in the human brain. These radioligands were stereospecifically prepared by electrophilic radiobromodestannylation of the respective tributylstannyl precursors using no-carrier-added [⁷⁶Br]BrNH₄ and peracetic acid. Preliminary pharmacological characterizations were determined by biodistribution, autoradiography, competition, displacement and metabolite studies in rats. The (-,-)-configuration presented important specific uptakes in brain muscarinic cholinergic receptor (mAChR)-rich structures and in heart, low metabolization rates and an apparent M₂ selectivity. The (-,+)-configuration revealed more rapid clearance, lower uptake, a higher metabolization rate and an apparent M₁ selectivity. Reversibility of the binding was confirmed for the three radiotracers. Positron emission tomography in the living baboon brain revealed high and rapid uptake in the brain and accumulation in the mAChR-rich structures studied. At 30 min p.i., theE(-,-)-radiotracer reached a plateau in cortex, pons and thalamus with concentrations of 29%, 24% and 19% ID/l, respectively.Z(-,-)-[⁷⁶Br]BrQNP also accumulated in these structures, reaching a maximal uptake (27% ID/l) in the cortex 2 h p.i. At 5 min p.i. a plateau (17% ID/l) was only observed in the cortex for theE(-,+)-[⁷⁶Br]BrQNP; by contrast, the other structures showed slow washout. After 3 weeks, the (-,-)-radiotracers were studied in the same baboon pretreated with dexetimide (1 mg/kg), a well-known muscarinic antagonist. In all the mAChR structures, the highly reduced uptake observed after this preloading step indicates that these radiotracers specifically bind to muscarinic receptors.Z(-,-)-[⁷⁶Br]BrQNP, which is displaced in higher amounts from M₂ mAChR-enriched structures, reveals an M₂ affinity. The two isomers having the (-,-)-configuration are potential probes for investigating central muscarinic receptors. The absolute configuration on the acetate chiral centre influences their muscarinic subtype selectivity and thecis-trans isomerism of the vinyl moiety affects their specific fixation.     

Systemic endotoxin and gastric mucosal pH are the best parameters to predict lethal outcome in a porcine model of abdominal sepsis according to multivariate analysis.

This study was devised to identify sepsis-relevant parameters that early and reliably predict a lethal outcome in intra-abdominal sepsis. In 18 Duroc pigs, peritonitis was induced through standardized gastrotomy. Twelve hours later the defect was oversewn and the abdominal cavity lavaged thoroughly. Sepsis relevant parameters were measured before initiating therapy, and 30 min later animals were extubated and observed for a period of 6 days under adequate analgesia with free access to water and food. All parameters were correlated with survival postoperatively. In the treatment group, 7 out of 18 pigs (39%) died within the observation period. Endotoxin level at 30 min after initiation of therapy [17.9 EU/mL (+/- 12.1) vs. 110.9 EU/mL (+/- 21); p <.001] and Delta pHi [0.015 (+/- 0.011) vs. -0.039 (+/- 0.013); p =.016] were identified as the two parameters with highest predictive power regarding mortality in a multivariate analysis. In conclusion measurement of endotoxin and gastric tonometry should gain wider clinical application in septic patients.     

Urination Frequency and Cystic Pressure Resistance After Fractionated whole or Partial Irradiation of the Rabbit Urinary Bladder

Urination frequency and cystic pressure resistance have been used as end-points to assess x-ray-induced changes of bladder function. Whole or half bladders of adult male rabbits were irradiated, caudally or cranially. The absorbed dose was 33 Gy, 36 Gy or 39 Gy, given in 5 daily fractions. Animals which received a whole bladder dose of 39 Gy or 36 Gy showed increased urination frequency and enhanced bladder pressure resistance during the whole follow-up time of 100 weeks, compared with the sham-irradiated controls. At half bladder irradiation, only the highest doses (39 Gy to the cranial part of the bladder and 39 Gy or 36 Gy to the caudal part) gave rise to a slight increase in frequency at about 20 weeks after exposure.     

Association of human polyomavirus JC with peripheral blood of immunoimpaired and healthy individuals

JC virus (JCV) infection is regularly asymptomatic in healthy individuals. In contrast, in immunocompromised individuals, highly activated virus replication may lead to PML. Peripheral blood cells (PBCs) are found to habor JCV DNA in healthy and diseased individuals and it is discussed that they might be responsible for dissemination of the virus to the central nervous system (CNS) during persistence. To better understand the role of JCV DNA in PBCs for persistent infection and pathogenesis, the authors characterized the extent of JCV infection in Ficoll-gradient purified blood cells (peripheral blood mononuclear cells [PBMCs]) of healthy and human immunodeficiency virus type 1 (HIV-1)-infected individuals. Virus activation in PBMCs from healthy JCV-infected individuals was found at a rate of 0% to 38% at low polymerase chain reaction (PCR) sensitivity. In progressive multifocal leukoencephalopathy (PML) patients, a stronger signal was found, indicating increased virus activation. JCV DNA was regularly detected in T and B lymphocytes and in monocytes at low levels. However, granulocytes were shown to be the predominant reservoir of JCV DNA harboring high copy numbers. Although the overall distribution of viral genomes holds true for the population studied, in the individual, a markedly changed pattern of distribution can be found.     

Selective visualization of the Fallopian tube with magnetic resonance imaging

At present, X-ray hysterosalpingography is used commonly as a screening method for testing Fallopian tube patency, but the results are often unreliable due to mucous plugs or muscular contractions. Selective catheterization of the tubes under X-ray control is feasible, but is rarely used due to exposure of young individuals aiming for pregnancy to a high ionizing dose. Here, a case is described of a patient whose Fallopian tubes were selectively catheterized and visualized three-dimensionally under contrast-enhanced magnetic resonance imaging (MRI) guidance using a high-viscous gadoteric acid solution (Dotarem). In this patient, bilateral peritubal adhesions caused a blockage of the fimbrial part of the tube leading to transuterine spilling of tubal fluid. Laparoscopy followed by bilateral salpingectomy was then performed, which confirmed the three-dimensional MRI images, and the excised specimens were examined histologically. The advantages of this novel technique include the avoidance of ionizing damage to the gonads and the potential for development of more elaborate interventional methods, such as ballooning and stenting. It is intended to develop contrast MRI further, both for improved non-invasive visualization and for manipulative technology of the Fallopian tubes.     

Role of 4-1BB in Allograft Rejection Mediated by CD8+ T Cells

Blockade of traditional costimulatory molecules fails to inhibit rejection in many models where CD8+ T cells are sufficient to mediate rejection. This observation demonstrates that in many settings CD8+ T cells are not dependent upon CD28 or CD154 signals to mediate rejection. 4-1BB (CD137) has been shown to be an important regulatory molecule for CD8+ T cells in a variety of nontransplant models. Here we show that blocking the 4-1BB pathway significantly inhibited rejection of intestinal allografts by CD8+ but not CD4+ T cells. This effect was associated with significantly decreased expression of the genes encoding TNFalpha and secondary lymphoid chemokine (SLC) within the spleens of recipient mice. Disruption of the 4-1BB pathway also impaired the priming of alloantigen-specific CD8+ T cells and the accumulation of recipient dendritic cells within the spleen. These data directly demonstrate an important role for 4-1BB in allograft rejection; particularly rejection mediated by CD8+ T cells. Our data suggest that in addition to providing a direct costimulatory signal to T cells, the 4-1BB pathway may regulate other important steps in the immune response such as the migration of T cells and dendritic cells.