No abstract

Enterovirus 70 (EV70) is the major etiological agent of acute hemorrhagic conjunctivitis (AHC). EV70 m.o.i.- (multiplicity of infection) and time-dependently induced apoptosis in human Chang's conjunctival (HCC) cells. UV- or heat-inactivated EV70 did not induce apoptosis. EV70-induced apoptosis was inhibited by cycloheximide and methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK), but not actinomycin D and guanidine.HCl (although guanidine.HCl inhibited the apoptosis induced by EV70 infection at 0.5 PFU/cell for 18 h). EV70 infection induced activation of caspase-3 and -7 and degradation of the constitutively activated caspase-6. EV70-induced apoptotic DNA ladders and activated caspase-3 and -7, correlated with virus release. Caspase inhibitor IX (Z-VD-FMK) inhibited EV70-induced apoptosis and virus release, but not intracellular viral production. The results suggest that infectious virus and the syntheses of viral proteins especially EV70 proteases, but not viral genome RNA, are required for caspase-3 and -7-mediated EV70-induced apoptosis, and that apoptosis through cell lysis promotes EV70 release from HCC cells.

Calreticulin (CRT) was isolated and identified as a protein in rabbit kidney RK(13) cells that binds the apoptogenic bacterial cell wall (BCW) components, muramyl dipeptide (MDP) and peptidoglycan (PG). Mannan-agarose purified RK(13) cell CRT (rCRT) selectively bound sepharose-immobilized L,D-MDP and PG, but not L,L-MDP or D,D-MDP. Purified rCRT and bovine CRT (bCRT) also bound free PG and L,D-MDP demonstrated in bioassays of RK(13) cell apoptosis. The results suggest that, in RK(13) cells, (a) CRT is a specific binding protein for both L,D-MDP and PG and (b) CRT binding L,D-MDP or PG is dependent on the stereoisomeric configuration of the dipeptide (L-alanyl-D-isoglutamine) moiety. In addition, the results also suggest that, in RK(13) cells, the binding of L,D-MDP, L,L-MDP, D,D-MDP, or PG to CRT correlates with their capacities of inducing apoptosis.

Immunization with uv-light inactivated EV70 elicits a classical humoral immune response in rabbits. The protective activity of serum in EV70-infected human conjunctival cells, but not lens cells, was increased by IFN-alpha. Adjuvant MDP-induced conjunctivitis, increased blood-conjunctival barrier (BCB) permeability and anti-EV70 neutralizing activity in tear of seropositive rabbits. The results suggest immunization with inactivated EV70 could provide systemic as well as ocular protection during natural EV70 infection.

The stimulus for caspase-mediated renal cell apoptosis in septic acute renal failure (ARF) is unclear. To demonstrate the nephrotoxic effects of bacterial cell wall components, the anti-cellular activity of bacterial muropeptides (muramyl dipeptides), peptidoglycans, and lipopolysaccharides was investigated in rabbit kidney cells. Changes in the cell membrane (APOPercentage™ dye uptake), caspase activities, and DNA degradation were quantified colorimetrically and using densitometric assays and their inhibition by caspase-specific and pan-caspase inhibitors was determined. The onset and levels of APOPercentage™ dye-positive rabbit kidney cells, caspase activities, and DNA degradation were closely associated. Specific caspase-1, -2, -3, -4, -8, -10, and -12 inhibitors reduced caspase-3 activity by ≥40%, but only caspase-3 and -8-specific inhibitors reduced apoptotic DNA levels. Pan-caspase inhibitor Q-VD-OPh was 10-fold more effective at inhibiting rabbit kidney cell death, caspase activation, and DNA degradation than caspase-family inhibitor Z-VAD-FMK. Apoptosis was inhibited effectively by both pan-caspase inhibitors when applied early during the stimulus-to-response period. Multiple initiator and effector caspases were activated suggesting extrinsic, intrinsic, and endoplasmic reticulum/stress apoptotic pathway stimulation in rabbit kidney cells treated with bacterial cell wall components. The results provide in vitro support for bacterial cell wall-induced apoptosis as a pathogenic mechanism of renal cell death in septic ARF and support the potential prophylactic use of pan-caspase inhibitors to suppress septic ARF.