Improving ability of RSV microneutralization assay to detect G-specific and cross-reactive neutralizing antibodies through immortalized cell line selection.

Respiratory syncytial virus (RSV) is a significant cause of bronchiolitis and pneumonia. Protection against RSV is associated with neutralizing antibodies against the fusion (F) and attachment (G) glycoproteins. Several RSV vaccine candidates are in development, but their immunogenicity is hard to compare due to the little-understood differences between multiple RSV neutralizing antibody assays used. Existing assays utilize primarily Vero or HEp-2 cells, but their ability to detect G-neutralizing antibodies or antibodies against specific RSV strains is unclear. In this work, we developed an RSV microneutralization assay (MNA) using unmodified RSV and immortalized cell line derived from human airway epithelial cells (A549). Performance of A549-, HEp-2- and Vero-based MNA was compared under the same assay conditions (fixed amount of virus and cells) with regards to detection of neutralizing antibodies against RSV A or B viruses, G-reactive neutralizing antibodies, and effect of complement. Our results indicate that A549 cells yield the highest MNA titers, particularly in the RSV A/A2 MNA, are least susceptible to complement-enhancing effect of neutralizing titer readout and are superior to Vero or HEp-2 MNA at recognizing G-reactive neutralizing antibodies when no complement is used. Vero cells, however, can be more consistent at recognizing neutralizing antibodies against multiple RSV strains. The choice of substrate cells thus affects the outcome of MNA, as some immortalized cells better support detection of broader range of neutralizing antibodies, while others facilitate detection of G-targeting neutralizing antibodies, a long-thought prerogative of primary airway epithelial cells.

Prevention of cataract in diabetic mice by topical pyruvate.

BACKGROUND: It has been previously reported that oral administration of sodium pyruvate inhibits oxidative stress and cataract formation in diabetic animals. With a view to exploring the clinical usefulness of these findings, this study examined its preventive effect when administered topically as an eye drop. METHODS: Diabetes was induced by intraperitoneal injections of streptozotocin. At the onset of diabetes, an eye drop preparation containing 2.5% sodium pyruvate was administered six times a day at 90-minute intervals. Treatment was continued for 6 weeks. Cataract formation was monitored ophthalmoscopically after mydriasis with 1% tropicamide eye drops. Subsequently, the treated and untreated diabetic animals and the age-matched normal controls were euthanized, their eyes enucleated, and the lenses isolated for biochemical assessment of protein glycation and glutathione levels. RESULTS: Treatment with pyruvate eye drops was found to be significantly effective in inhibiting protein glycation. Glutathione levels were also better maintained. In addition, ophthalmoscopic examination revealed that the incidence of cataract in the pyruvate-treated group was only 12% as compared with the untreated diabetics in whom the incidence was 73%. Cataracts at this stage were largely equatorial. CONCLUSION: The results demonstrate that topical application of pyruvate can potentially be useful in attenuating or preventing cataract formation induced by diabetes and other conditions of oxidative stress.

Inhibition of glycolysis in the retina by oxidative stress: prevention by pyruvate.

Intraocular generation of reactive oxygen species (ROS) with consequent oxidative stress has been shown to be a significant factor in the pathogenesis of many vision-impairing diseases such as cataracts and retinal degenerations. Previous studies have shown that pyruvate can inhibit such oxidative stress. This is attributable to its property of scavenging various ROS and consequently inhibiting many of the apparent toxic reactions such as lipid peroxidation and loss of tissue thiols. It is hence expected that ROS will have an adverse effect on tissue metabolism also. The present investigations were hence undertaken to study the possibility that while scavenging ROS, the compound could be effective also in preventing the inhibition of tissue metabolism as well. Since glycolysis constitutes the major bioenergetic source of the retina, the objective of the present studies was to ascertain if the effects of pyruvate are indeed reflected in the maintenance of this pathway even when the tissue is exposed to ROS. This hypothesis was examined by incubating retinal explants in ROS-generating medium in the absence and presence of pyruvate and measuring (3)H(2)O generated from 5-(3)H glucose. In addition, the lactate generated was also measured. As hypothesized, ROS-induced inhibition of glycolysis indexed by the decrease in (3)H(2)O as well as lactate formation was significantly prevented by pyruvate. This effect was also reflected by the elevation of NAD/NADH ratio, a major pacemaker of glycolysis.

Oxidative stress in lens in vivo: inhibitory effect of caffeine. A preliminary report.

PURPOSE: Experiments have been conducted to study the hypothesis that caffeine would inhibit reactive oxygen species induced oxidative stress in the lens in vivo, with implications of attenuating or preventing cataract formation. METHODS: Oxidative stress was directly induced by administering 24% galactose diet to young adult rats. The treated group was fed a diet containing 24% galactose + 1% caffeine. Oxidative stress inflicted to the lens was assessed by measurement of glutathione (GSH) depletion and observing the status of lens clarity. RESULTS: Caffeine administration was found to minimize the loss of GSH. This was also associated with a better maintenance of lens transparency as compared to the untreated galactosemic group. CONCLUSIONS: The studies demonstrate that caffeine could be helpful in inhibiting oxidative stress in the lens with the consequence of attenuating cataract formation.

Intraocular penetration of pyruvate following its topical administration in mice.

Previous studies from our laboratory have demonstrated that pyruvate, an endogenous alpha-keto acid metabolite, has a protective effect against oxidative stress induced damage to the ocular tissues including the lens, in which in addition to exerting its protective effect against tissue damage caused by oxyradicals generated under organ culture, it is also found effective in preventing actual cataract formation in vivo in animal models undergoing direct oxidative stress as well as in diabetes. In the latter studies, pyruvate was administered mixed with diet and drinking water. However, with the view of the desirability of treating eye diseases by topical administration of the pharmacological agents, the present studies were conducted to determine the penetrability of pyruvate through the cornea to the aqueous humor and the lens following its topical administration as its ester, ethyl pyruvate (EP). These experiments were done in CD-1 mice. After instillation of the drops in the conjunctival cul-de-sac, aqueous humor samples were aspirated at the desired times and analyzed for pyruvate. In a separate group of animals, analyses were done also in the lens. Analyses were done spectrophotometrically by monitoring the decrease in absorption of NADH due to the reduction of pyruvate to lactate by lactate dehydrogenase. The levels of pyruvate were found to be significantly elevated in both the aqueous humor as well as the lens, the peak concentrations being 4.7 and 3.6 mM, respectively. Such levels have been previously shown to be effective in exerting its antioxidant effects. The results are therefore considered pharmacological significant from the point of view of its potential use for topical treatment of cataracts induced by oxidative stress and diabetes.

Oxidative damage to lens in culture: reversibility by pyruvate and ethyl pyruvate.

It is generally believed that prophylactic intake of antioxidants is beneficial in delaying the onset of some aging manifestations such as cataract. However, whether such a supplementation will also be effective if the pathophysiological process has already set in remains a largely open question. We examined this possibility with lens changes leading to cataract formation, since cataract genesis is intimately related to a continued generation of reactive oxygen species (ROS) in the aqueous humor. Since the formation of cataract is a well-defined progressive disease, starting with an early refractive change and leading to gradual enhancement of opacification, we hypothesized that even a belated start with an appropriate anti-oxidant could halt the pathological process and delay cataract maturation and vision impairment. Using lens cultures, we tested this hypothesis with pyruvate, known to be an effective and highly potent ROS scavenger. Adding pyruvate to the culture medium after lenses had sustained a 50% damage was significantly effective in preventing progress. This was apparent by better maintenance of the active rubidium transport activity in these lenses compared to controls without pyruvate treatment. Glutathione levels were also higher in the pyruvate group.

Attenuation and delay of diabetic cataracts by antioxidants: effectiveness of pyruvate after onset of cataract.

Cataract is one of the most significant vision-impairing complications of diabetes. The present study examined the feasibility of inhibiting cataract formation by treatment with pyruvate, a metabolite known to effectively scavenge reactive species of oxygen and inhibit protein glycation, both known to be involved in the genesis of diabetic cataracts. In addition, pyruvate stimulates tissue metabolism, which is depressed with the onset of cataract formation. The objective of our experiments was to determine if this compound could be effective in offsetting the progress of cataract, specifically if administered after the diabetes-induced lens changes have begun, as opposed to the previous reports wherein it has been reported to delay cataract formation if administered prophylactically with the immediate onset of diabetes. Diabetes was induced by intraperitoneal administration of streptozotocin to mice. Lens transparency was assessed by slit lamp examination and its photography. ATP was determined enzymatically by reacting it with luciferin-luciferase mixture and measuring the fluorescence intensity. The findings described herein are in accordance with this possibility. The incidence of cataract in the group of diabetic animals, where treatment with pyruvate was initiated after the initial lens changes set in, was significantly lower at all times of observation in comparison to the untreated diabetic group. In addition, the severity of opacities in the pyruvate-treated group, when present, was much minor, the transparency of these cases being close to that in the control animals. The ophthalmic findings are supported biochemically by ATP levels, which were significantly higher in the pyruvate group in comparison to the untreated group. The present findings emphasize the clinical usefulness of initiating treatment with anti-oxidants and metabolic agonists even when the lens changes are detected at the time of the diabetes diagnosis. The latter usually comes much later than the onset of visual aberrations. Prophylaxis is not an absolute requirement.

[Participation of midbrain central gray neurons in the organization of defensive conditioned reflexes in the cat]. / Uchastie neironov tsentral'nogo serogo veshchestva srednego mozga v organizatsii oboronitel'nogo uslovnogo refleksa koshki.

Neuronal reactions of the periaqueductal gray matter were studied in cats during conditioning defence reflex to auditory stimuli. Phasic and tonic spike responses lasting up to 3s developed in the recorded neurons in response to conditional stimulus. These reactions had developed long before the elaboration of the conditioned reflex and disappeared significantly later than the conditioned reflex became extinct. The generation of the spike response after elaboration of the conditioned reflex took place 100-200 ms before the appearance of the movement evoked by the conditional stimulus. An increase of the tonic discharge of the recorded neurons also preceded the voluntary movements by 100-500 ms. Neuronal responses in the periaqueductal gray matter to differential stimuli could be elaborated with great difficulty; they had the form of group discharges 150-200 ms long. These responses were unstable. On the basis of the obtained data the conditioned reflex is considered as a result of a multilevel hierarchic process of neuronal activity change which begins in unspecific structures of the mesencephalon.

[Effect of a new cholinesterase reactivator, diethixime, on the central nervous system]. / Deistvie novogo reaktivatora kholinesterazy diétiksima na tsentral'nuiu nervnuiu sistemu

A new cholinesteras reactivator--chlorohydrate of S-diethylaminoethyl ether p-bromo benzoylthiohydroxime acid (diethixime), containing a tertiary nitrogen atom in the molecule, was shown to produce a central effect in a dose of 20 mg/kg--1/50 LD50--in contrast to diproxime in a dose of 3 mg/kg, containing a quarternary nitrogen atom, under intoxication of albino rats and rabbits with dimethyl-dichlorynylphosphate. This effect was confirmed by the restortion of the cholinesterase activity in different parts of the rabbit brain, by the normalization of the EEGand of the functional stateof motor neurons of the rat spinal cord.