Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection

A unique coronavirus severe acute respiratory syndrome-coronavirus (SARS-CoV) was revealed to be a causative agent of a life-threatening SARS. Although this virus grows in a variety of tissues that express its receptor, the mechanism of the severe respiratory illness caused by this virus is not well understood. Here, we report a possible mechanism for the extensive damage seen in the major target organs for this disease. A recent study of the cell entry mechanism of SARS-CoV reveals that it takes an endosomal pathway. We found that proteases such as trypsin and thermolysin enabled SARS-CoV adsorbed onto the cell surface to enter cells directly from that site. This finding shows that SARS-CoV has the potential to take two distinct pathways for cell entry, depending on the presence of proteases in the environment. Moreover, the protease-mediated entry facilitated a 100- to 1,000-fold higher efficient infection than did the endosomal pathway used in the absence of proteases. These results suggest that the proteases produced in the lungs by inflammatory cells are responsible for high multiplication of SARS-CoV, which results in severe lung tissue damage. Likewise, elastase, a major protease produced in the lungs during inflammation, also enhanced SARS-CoV infection in cultured cells

Differences in Antibody Responses of Individuals with Natural Infection and Those Vaccinated against Pandemic H1N1 2009 Influenza▿

The differential antibody response measured by the commonly used hemagglutination inhibition (HI) and microneutralization (MN) assays in patients with natural infection and vaccination has not been fully assessed. HI and conventional MN (CMN) assays were performed on sera from 651 patients with natural infection by pandemic H1N1 2009 influenza virus and on sera from 567 recipients of the corresponding vaccine. Surprisingly, the overall seroprotection rates determined by CMN and HI assays in vaccine recipients were only 44.8 and 35.1%, respectively. Antibody titers measured by the CMN assay was significantly higher than that obtained by HI assay in vaccine recipients aged ≥50 years, but these titers were not significantly different among younger vaccine recipients. In contrast, the HI titer was greater than the CMN titer for the age group from 16 to 29 years but was not significantly different in other age groups for natural infection. Lower antibody levels were found in both naturally infected patients and immunized recipients in the older than in the younger age groups, but naturally infected patients exhibited higher HI and CMN titers than did the corresponding vaccine recipients. In addition, we developed a rapid fluorescent focus microneutralization (FFMN) assay to test sera from naturally infected patients. The FFMN assay has a better correlation with CMN than with HI (ρ = 0.810 versus 0.684), which is expected of neutralizing antibody mainly targeted toward the inhibition of viral entry into cells. The higher antibody level elicited by natural infection than by vaccination may be related to differences between antigen presentation by the intramuscular route of vaccination and mucosal viral replication in mucosal cells of the respiratory tract

Evaluation of pharmacological activities and assessment of intraocular penetration of an ayurvedic polyherbal eye drop (Itone™) in experimental models

<p>Abstract</p> <p>Background</p> <p>The polyherbal eye drop (Itone™) is a mixture of aqueous distillates of nineteen traditionally used ingredients that sum up to impart potency to the formulation and make it a useful adjunct in various ocular pathologies. However, as there have been no controlled experimental studies accounting to the above claim, therefore, the present study was designed to evaluate the polyherbal formulation (PHF) for antiangiogenic, anti-inflammatory, anticataract, antioxidant and cytotoxicity in addition to the evaluation of intraocular penetration of PHF in rabbit eyes using LC-MS/MS.</p> <p>Materials and methods</p> <p>Antiangiogenic activity of the PHF was evaluated using <it>in ovo</it> chick chorio-allantoic membrane (CAM) assay and <it>in vivo</it> cautery induced corneal neovascularization assay in rats. Anticataract potential was evaluated using steroid induced cataract in developing chick embryos, sodium selenite induced cataract in rat pups and galactose induced cataract in rats. The antioxidant activity was evaluated using di-phenyl picryl hydrazyl (DPPH) radical scavenging assay. Anti-inflammatory activity was evaluated <it>in vitro</it> using inhibition of LTB₄ formation in human WBCs and <it>in vivo</it> using carrageenan induced paw edema assay in rats. The cytotoxicity was evaluated against HeLa cancer cell lines using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore evaluation of the intraocular penetration of the PHF was carried out in rabbit eyes via aqueous humor paracentesis and further analysis using LC-MS/MS.</p> <p>Results</p> <p>PHF significantly inhibited VEGF induced proliferation of new blood vessels in CAM assay and inhibited the cautery induced corneal neovascularization in rats. Additionally, PHF showed noticeable delay in the progression of cataract in the selenite and galactose induced cataract models whereby the PHF treated lenses were graded for stages II and III respectively. However, the PHF did not show any anticataract activity in the hydrocortisone induced cataract model. Moreover, PHF exhibited anti-inflammatory activity whereby it showed 39.34% inhibition of LTB₄ formation and significantly inhibited carrageenan induced paw edema in rats. Eight compounds of PHF viz. camphor, casticin, curcumin-II, quercetin, rosmarinic acid, γ-terpinene, β-pinene and dipentene exhibited transcorneal penetration in rabbit eyes.</p> <p>Conclusion</p> <p>The significant antiangiogenic and anti-inflammatory activities evinced by the PHF merits further investigation for ocular neovascular and inflammatory diseases in humans.</p