Novel immunofluorescence assay using recombinant nucleocapsid-spike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a "gold standard" during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chng, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422, 2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.     

Immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is a novel infectious disease caused by the SARS-associated coronavirus (SARS-CoV). There are four major structural proteins in the SARS-CoV, including the nucleocapsid, spike, membrane, and small envelope proteins. In this study, two sets of truncated fragments of spike protein were generated, the first were approximately 210-bp nonoverlapping fragments and the second were overlapping segments of 750 to 900 bp. From these 23 fragments, we identified a fragment of 259 amino acids (amino acids 441 to 700) that is a major immunodominant epitope. This fragment was highly expressed, and the purified fragment C could detect all 33 SARS patient serum samples tested, collected from 7 to 60 days after the onset of fever, but had no reactivity with all 66 healthy human serum samples tested. Thus, fragment C of spike protein was identified as an immunodominant antigen and could be used for serological detection of SARS-CoV infection.     

Retinoids and skin: microarrays shed new light on chemopreventive action of all-trans retinoic acid

Despite the use of retinoids in the clinic for many years, their mode of action in the prevention of skin cancer is still unclear. Recent microarray analyses of the chemopreventive effect of all-trans retinoic acid (ATRA), one of the primary naturally occurring biologically active retinoids, in the two-stage mouse skin chemical carcinogenesis model have provided novel insight into their action. Comparison of the gene expression profiles of control skin to skin subjected to the two-stage protocol for 3 wk, with or without ATRA, has shown that approximately half of the genes regulated by 12-o-tetradecanoylphorbol-13-acetate (TPA) are oppositely regulated when ATRA is coadministered with TPA. It was further shown the Raf/Mek/Erk branch of mitogen-activated protein (MAP) kinase pathway contains a disproportionate number of oppositely regulated genes, thereby implicating it as one of the key pathways involved in tumor promotion by TPA, that is blocked by ATRA. This result has pointed the way toward the detailed study of Raf/Mek/Erk pathway signaling in skin cancer development and its potential as a target pathway for chemoprevention by ATRA and other chemopreventive drugs.     

Influence of nanosized delivery systems with benzyl nicotinate and penetration enhancers on skin oxygenation

Many novel nanosized delivery systems have been designed for topical application of drugs since they can overcome the skin barrier and improve drug bioavailability. The increased absorption is often a consequence of a reversibly disrupted barrier function of the skin by the vehicle itself or by specific ingredients that act as penetration enhancers. This paper reports the effects of two nanosized systems (microemulsion and liposomes), in the presence and absence of penetration enhancers (PE), on the topical delivery of a lipophilic drug in vivo and compares that to classical hydrogel formulation. A vasodilator benzyl nicotinate (BN), which increases the blood flow of the skin, was incorporated into the formulations, and skin oxygenation was followed by electron paramagnetic resonance oximetry. It was found that microemulsions and liposomes (with or without PE) accelerate the rate of BN action when compared to hydrogel. However, incorporation of PE in microemulsion also improves the effectiveness of BN action. To understand why PE enhances the action of BN, its effect on the structure of the stratum corneum was investigated in vitro. The increased fluidity of the stratum corneum lipids provides an explanation for the greater penetration of BN into the skin when the drug and PE are together incorporated into the appropriate formulation.     

Gastric cancer in women: A regional health-center seven year retrospective study

AIM: To investigate whether regional geography influences ethnic and gender trends for the development of gastric cancer(GC).METHODS: This retrospective analysis of the INVISION patient database at Louisiana State University Health Sciences Center-Shreveport(LSUHSC-S), a southern United States regional hospital, was performed from 2005-2011. Using the international statistical classification of diseases 9(ICD-9), inpatient, day surgery outpatient, and emergency outpatient diagnosis codes entered into medical records were used to identify GC patients. For each study year, the patients were evaluated for age, ethnicity, and gender, and each patient was counted only once throughout the study. Subsequent patient encounters were counted as visits and separated by inpatient and clinic visits. Complex or severe disease may require more frequent and intensive clinical management; therefore, we evaluated annual clinic visits as "surrogate markers" of disease severity. Finally, we studied the primary diagnosis for Helicobacter pylori(H. pylori) infection(ICD-9 code 41.86) as an additional factor that might increase the risk of GC.RESULTS: A total of 285 patients were diagnosed with GC at LSUHSC-S between 2005 and 2011. African Americans(181 patients, 89 males and 92 females, 63.5% of total patients) had significantly higher frequencies of GC diagnosis compared with non-Hispanic whites(104 patients, 54 males and 50 females, 36.5% of total patients), at a ratio of 1.74(P = 0.002). Within each ethnic group, men and women were diagnosed at approximately equal annual rates. Our findings differed significantly from United States national trends, which found that African American females and white females had lower risks for GC than their corresponding male counterparts. The United States national trend between 2005 and 2011 showed that African Americans males had a higher incidence of GC, with an annual mean(per 100000) of 16.31 ± 0.76 compared with white males(9 ± 0.1, P 0.001), African American females(8.7 ± 0.34, P 0.001) and white females(4.05 ± 0.07, P 0.001). Among the GC patients, the number of clinic visits was highest among African American males(195.1 ± 28.1), who had significantly more clinic visits than African Americans females(123 ± 13.02, P 0.05), white males(41.57 ± 4.74, P 0.001) and white females(35 ± 8.9, P 0.001). Similar trends were found for inpatient visits, with an annual mean of 11.43 ± 1.5 forAfrican American males, followed by African American females(7.29 ± 1.36), white males(2.57 ± 0.69) and white females(1.57 ± 0.612). African American males had significantly more inpatient visits than white males(P 0.001), and African American females had more inpatient visits than white females(P 0.01). African American patients showed the highest frequency of H. pylori positive status, with approximately 72% vs 28% for the white patients. CONCLUSION: Increase in GC diagnoses among women at LSUHSC-S is significantly higher than United States national averages, suggesting local geographic and socioeconomic influences may alter GC disease course.     

A 3D co-culture of three human cell lines to model the inflamed intestinal mucosa for safety testing of nanomaterials

Oral exposure to nanomaterials is a current concern, asking for innovative biological test systems to assess their safety, especially also in conditions of inflammatory disorders. Aim of this study was to develop a 3D intestinal model, consisting of Caco-2 cells and two human immune cell lines, suitable to assess nanomaterial toxicity, in either healthy or diseased conditions. Human macrophages (THP-1) and human dendritic cells (MUTZ-3) were embedded in a collagen scaffold and seeded on the apical side of transwell inserts. Caco-2 cells were seeded on top of this layer, forming a 3D model of the intestinal mucosa. Toxicity of engineered nanoparticles (NM101 TiO₂, NM300 Ag, Au) was evaluated in non-inflamed and inflamed co-cultures, and also compared to non-inflamed Caco-2 monocultures. Inflammation was elicited by IL-1β, and interactions with engineered NPs were addressed by different endpoints. The 3D co-culture showed well preserved ultrastructure and significant barrier properties. Ag NPs were found to be more toxic than TiO₂ or Au NPs. But once inflamed with IL-1β, the co-cultures released higher amounts of IL-8 compared to Caco-2 monocultures. However, the cytotoxicity of Ag NPs was higher in Caco-2 monocultures than in 3D co-cultures. The naturally higher IL-8 production in the co-cultures was enhanced even further by the Ag NPs. This study shows that it is possible to mimic inflamed conditions in a 3D co-culture model of the intestinal mucosa. The fact that it is based on three easily available human cell lines makes this model valuable to study the safety of nanomaterials in the context of inflammation.