Adenovirus-mediated interleukin-10 gene transfer inhibits post-transplant fibrous airway obliteration in an animal model of bronchiolitis obliterans

Bronchiolitis obliterans, a form of chronic allograft rejection characterized by progressive fibrous obliteration of the airways, is the major obstacle limiting prolonged survival of lung transplant recipients. To date, no effective therapy against this fatal complication exists. Interleukin-10 (IL-10), an anti-inflammatory and immunosuppressive cytokine, inhibits various T cell and antigen-presenting cell functions. We examined the effect of IL-10 in an animal model for bronchiolitis obliterans. A heterotopic tracheal transplant model was used. IL-10 was administered to the recipient either in its recombinant form by osmotic minipump or by adenoviral-mediated IL-10 gene transfer (Ad5E1mIL-10). Successful gene transfection and expression was confirmed by measuring circulating IL-10 protein. Tracheal allografts were assessed histologically based on a scoring system. IL-10 administration (in recombinant form or by gene transfer) inhibited the development of fibrous airway obliteration 3 weeks after transplantation in comparison to untreated controls (p < 0.05). This was demonstrated only if the delivery was initiated 5 days after transplantation and not if it was started at the time of transplantation. A single administration of the gene construct was sufficient to achieve the desired effect. We have shown that IL-10 can prevent the development of airway fibro-obliteration in this model. Gene transfection at a site distant from a graft can be used to produce a desired effect on the graft. IL-10 may be of major importance in the control of post-transplant bronchiolitis obliterans. The timing of its administration is critical and further studies are required to determine the mechanisms underlying the observed effects of IL-10.     

ATP-dependent proteolysis in mitochondria. m-AAA protease and PIM1 protease exert overlapping substrate specificities and cooperate with the mtHsp70 system

To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at non-permissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and the m-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.     

Vaccinia DNA topoisomerase I: evidence supporting a free rotation mechanism for DNA supercoil relaxation

The Vaccinia type I topoisomerase catalyzes site-specific DNA strand cleavage and religation by forming a transient phosphotyrosyl linkage between the DNA and Tyr-274, resulting in the release of DNA supercoils. For type I topoisomerases, two mechanisms have been proposed for supercoil release: (I) a coupled mechanism termed strand passage, in which a single supercoil is removed per cleavage/religation cycle, resulting in multiple topoisomer intermediates and late product formation, or (2) an uncoupled mechanism termed free rotation, where multiple supercoils are removed per cleavage/religation cycle, resulting in few intermediates and early product formation. To determine the mechanism, single-turnover experiments were done with supercoiled plasmid DNA under conditions in which the topoisomerase cleaves predominantly at a single site per DNA molecule. The concentrations of supercoiled substrate, intermediate topoisomers, and relaxed product vs time were measured by fluorescence imaging, and the rate constants for their interconversion were determined by kinetic simulation. Few intermediates and early product formation were observed. From these data, the rate constants for cleavage (0.3 s(-1)), religation (4 s(-1)), and the cleavage equilibrium constant on the enzyme (0.075) at 22 degrees C are in reasonable agreement with those obtained with small oligonucleotide substrates, while the rotation rate of the cleaved DNA strand is fast (approximately 20 rotations/s). Thus, the average number of supercoils removed for each cleavage event greatly exceeds unity (delta n = 5) and depends on kinetic competition between religation and supercoil release, establishing a free rotation mechanism. This free rotation mechanism for a type I topoisomerase differs from the strand passage mechanism proposed for the type II enzymes.     

Autonomic dysfunction as the presenting feature of Guillain-Barré syndrome

Autonomic dysfunction has been demonstrated in various conditions associated with peripheral neuropathy such as acute intermittent porphyria, amyloidosis, and Guillain-Barré syndrome (GBS). In the latter, hypertension is an associated complication that typically occurs after neurological signs are already present. We report a case of a patient with autonomic dysfunction as the presenting feature who was admitted to the coronary unit with chest pain and hypertension. Subsequently, he developed progressive symmetric muscle, weakness, sensory changes, and areflexia. GBS was then diagnosed based on the clinical picture, albuminocytologic dissociation in the cerebrospinal fluid, and electrodiagnostic abnormalities suggestive of demyelinative polyneuropathy with conduction block. Few cases in the literature have reported autonomic dysfunction as the presenting feature of GBS, such as in this case. In a previously asymptomatic patient, acute onset of autonomic dysfunction should alert the physician to the possibility of an acute polyneuropathy, such as GBS.     

A five year follow up into changes in caries experience amongst a sample of 12 year old children from Athens

The airway functions in pregnancy have been widely studied but reports obtained from Western and Indian population show divergence. While the Indian populations show significant changes in total and timed vital capacity (FVC and FEV1), the Western counterparts dismiss such changes as insignificant. Our results show insignificant alteration in airway function and support the results reported for Western population.     

Development of a method for direct quantification of cytomegalovirus antigenemia by flow cytometry

Cytomegalovirus (CMV) antigenemia was directly detected in polymorphonuclear leukocytes (PMNLs) from transplant recipients by using flow cytometry (FC). Two fixation and permeabilization methods and seven anti-CMV monoclonal antibodies (MAbs) were evaluated. 1C3, SL20, and NEA-9221 MAbs were more efficacious. The antigenemia detection threshold of FC was 0.05% positive PMNLs, and percentages correlated well with DNA viral load and the appearance of clinical symptoms.