Adenoviral-expressed GP<Subscript>5</Subscript> of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions

Summary.  The ORFs 5, 6 and 7, encoding for the three major structural proteins, GP₅, M and N, of the IAF-Klop strain of PRRSV were cloned and expressed in 293 cells using replication-defective human type 5 adenoviral vectors (hAdVs). Although the M protein gene could be cloned into hAdVs and expressed constituvely in 293 cells under the control of the hCMV immediate early promotor/enhancer, hAdVs expressing N and GP₅ proteins, which appeared to be toxic or interfered with adenovirus replication, could only be generated by inclusion of a tetracycline-regulatable promotor in the transfer vector pAdTR5. The recombinant (rec) proteins appeared similar to the authentic viral proteins in regards to their M _rs and antigenicities. However, the recGP₅ apparently possesses different N-linked oligosaccharides residues. Its sensitivity to endo-β-galactosidase digestion indicates that poly-N-acetyllactosamine is present on the individually-expressed protein, but not on the authentic GP₅ anchored into the virion envelope. The recGP₅ apparently accumulates within the ER compartment as a glycoprotein that possesses high-mannose N-linked oligosaccharide side chains sensitive to endo-β-N-acetylglucosaminidase H treatment, by contrast to its viral counterpart for which N-linked oligosaccharide side chains are of both high-mannose and complex types. Coinfection of 293 cells with hAdVs expressing the M and GP₅ did not lead to M-GP₅ heterodimer formation, as demonstrated in PRRSV-infected cells. Moreover, cells infected with inducible hAdV/ORF5 showed that GP₅ of the North American strain is proapoptotic. Indeed, when the expression cassette was turned-on, caspase 3 activity in hAdV/ORF5 infected cells was enhanced and DNA fragmentation could be detected by TUNEL assays. Pigs intradermally injected twice with hAdV/ORF5 developed antibody titers to the authentic viral GP₅ as soon as 10 days following challenge with the homologous virulent PRRSV strain, as revealed by Western blot and virus neutralization tests, suggesting the establishment of a specific immune memory. In memoriam. The authors wish to offer their condolences to the family and friends of Dr. Serge Dea, who died on January 3, 2003, and acknowledge his tremendous accomplishment in the field of Nidoviruses. Received April 8, 2002; accepted October 15, 2002 Published online March 3, 2003     

Therapeutic angiogenesis using autologous bone marrow stromal cells: improved blood flow in a chronic limb ischemia model

BACKGROUND: We evaluated the effect of autologous marrow stromal cells (MSCs) on neovascularization and blood flow in an animal model of chronic limb ischemia. METHODS: Chronic hind limb ischemia was created by ligating the left common iliac artery of male Lewis rats. Three weeks after ligation, 5.0 million LacZ+MSCs (n = 10) or culture medium (n = 10) were injected into the anteromedial muscle compartment of the left thigh. At 4 and 6 weeks after injection, half the animals (n = 5) from each group underwent femoral artery ultrasonic blood flow measurements of the ischemic and nonischemic limbs to obtain a flow ratio. The animals also underwent angiography and measurements of blood vessel density and arteriolar density. Qualitative histologic assessment of the limb muscles was performed. RESULTS: LacZ+MSCs were found to differentiate into endothelium (F VIII+), vascular smooth muscle (positive a-smooth muscle actin), skeletal muscle (positive desmin), and adipocytes. Ischemic hind limbs where MSCs were implanted had greater vascular density and arteriolar density than control limbs (p < 0.001). Femoral artery flow index (left femoral artery flow/right femoral artery flow) was 0.89 +/- 0.12 and 0.90 +/- 0.06 for rats injected with MSCs measured at 4- and 6-weeks, respectively, compared with 0.50 +/- 0.15 and 0.50 +/- 0.10 for the control rats (p < 0.001). Angiography demonstrated reconstitution of the left femoral artery in rats that received MSC implantation through pelvic and abdominal wall collateral formation. CONCLUSIONS: Local MSC implantation induces a neovascular response resulting in a significant increase in blood flow to the ischemic limb. Marrow stromal cells are also capable of spontaneously regenerating the various components of muscular tissues.     

The photopic ERG luminance-response function (photopic hill): method of analysis and clinical application

With progressively brighter stimuli, the amplitude of the photopic b-wave first increases, briefly saturates and then decreases gradually to reach a plateau, where the amplitude of the b-wave equals that of the a-wave; a phenomenon previously presented as the photopic hill. The unique presentation of this luminance-response function seriously complicates its analysis with curve fitting equations such as that of Naka-Rushton used for scotopic electroretinogram. We report a method of analysis of the photopic hill based on easily identifiable and reproducible features of the ascending and descending limbs of this function. The clinical usefulness of these parameters is illustrated with selected cases of retinal disorders.     

Graded contribution of retinal maturation to the development of oxygen-induced retinopathy in rats

PURPOSE: Newborn rats exposed to hyperoxia during the first days of life have been shown to exhibit not only vasculopathy but also permanent changes in the structure and function of the retina. Given that the rat retina is immature at birth and that the maturation process continues until the opening of the eyes at 14 days of life, this study was conducted to investigate the susceptibility of the retina to oxygen toxicity as a function of the degree of retinal maturity reached at the time of oxygen exposure. METHODS: Newborn rats were exposed to hyperoxia during selected postnatal day intervals. Scotopic electroretinograms were recorded at 30 and 60 days of age, and retinal histology was obtained at the end of the study. RESULTS: There was a strong correlation between the duration of the hyperoxic event and the structural and functional consequences in the retina. However, the repercussions were significantly more profound when the exposure to oxygen occurred within the second week of life (6-14 days), compared with earlier (0-6 days) or later periods (14-28 days). CONCLUSIONS: The results strongly suggest that the structural and functional retinal changes secondary to postnatal hyperoxia are not only the direct consequence of exposure to high levels of oxygen (i.e., free radicals), but also are determined by the level of retinal maturity reached at the time of oxygen exposure. The results also indicate that the structural anomalies precede the functional impairments.     

Intracerebral transplantation of oligodendrocytes in mice

We describe in this paper experiments in which oligodendrocytes (from newborn mouse, human embryonic brain, or isolated from adult rat brain) have been transplanted into the brain of the newborn mouse. Experimental conditions (Shiverer model) allowed the detection of myelin formed by transplanted oligodendrocytes into the Shiverer brain. The transplanted oligodendrocytes have been shown to survive, migrate over long distances and myelinate host axons. The maturation of transplanted oligodendrocytes depends upon the age of the brain tissue in which they differentiate.     

Temporal relationship between the ERG and geniculate unit activity in rabbit: influence of background luminance.

The comparative analysis of the retinal and lateral geniculate nucleus (LGN) intensity-response function revealed that the timing of the LGN unit response was highly correlated to that of one oscillatory potential (OP2). To examine if this OP2-LGN intensity-response function was retained irrespective of the state of retinal adaptation, we performed simultaneous recordings to the ERG and single-cell unit activity at the geniculate level evoked to a flash of constant energy while the level of background luminence was varied. For a stimulus of 6.7 cd m-2 sec, the shifts in latency induced by increasing the background luminance from 0 to 125 cd m-2 are of 1.17 +/- 0.61 msec for OP2, a value almost identical (P greater than 0.10; n = 36) to the one obtained at the geniculate level (1.11 +/- 0.88 msec). However, when a dimmer flash is used, the latency shifts are not so well correlated. The latter could be partly explained by the threshold nature of the resulting stimulus (i.e. high photopic background combined with a dim flash).     

Congenital Erosive and Vesicular Dermatosis with Reticulated Scarring in a Newborn: An Innovative Treatment Using a Silicone Dressing

Abstract: Congenital erosive and vesicular dermatosis healing with reticulated supple scarring is a rare entity presenting in the newborn with crusted erosions and vesicles that heal relatively rapidly, forming unique reticulated scars. We report the case of a premature baby 31 weeks old. Diagnosis was confirmed by skin biopsies, and the clinical improvement was excellent, with complete healing observed within 7 weeks. This report highlights clinical and histopathologic features, and a new successful treatment approach using a silicone dressing.     

Optic nerve blockade influences the retinal responses to flash in rabbits

The presence of retinopetal fibers in mammals has been debated many times in the past two decades. Do rabbits have a retinopetal system? This question is addressed with the present investigations. In anesthetized and paralysed rabbits the b-wave and the oscillatory potentials (OP) are recorded at the cornea. The optic nerve is isolated retrobulbarly and is gently hooked to a curved injecting capillary. Through the latter lidocaine hydrochloride is pressure injected. This drug interrupts the neuronal flow travelling along the nerve. A steel electrode is positioned in the optic chiasm allowing us to monitor the evoked field potentials from the tested and untested eyes. The optic nerve blockade produces the following observations: (1) the amplitude of the b-wave is not significantly altered; and (2) the amplitudes of the long latency OP are significantly increased. The retinal capacity to respond to a second flash after the application of an initial light pulse was evaluated by varying the interval between the two flashes. After optic nerve blockade the recovery of the retinal responsiveness is considerably slower. Fourier analysis indicated that the highest power increases occurred around 200 Hz. It is difficult to escape the suggestion that rabbits possess retinopetal fibers.