Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription͞translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immuneresponse profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naïve humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naïve mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.pox virus ͉ proteomics ͉ vaccine ͉ Francisella tularensis

Pemphigus vulgaris (PV) is a mucocutaneous blistering disease characterized by IgG autoantibodies against the stratified squamous epithelium. Current understanding of PV pathophysiology does not explain the mechanism of acantholysis in patients lacking desmoglein antibodies, which justifies a search for novel targets of pemphigus autoimmunity. We tested 264 pemphigus and 138 normal control sera on the multiplexed protein array platform containing 701 human genes encompassing many known keratinocyte cell-surface molecules and members of protein families targeted by organ-non-specific PV antibodies. The top 10 antigens recognized by the majority of test patients’ sera were proteins encoded by the DSC1, DSC3, ATP2C1, PKP3, CHRM3, COL21A1, ANXA8L1, CD88 and CHRNE genes. The most common combinations of target antigens included at least one of the adhesion molecules DSC1, DSC3 or PKP3 and/or the acetylcholine receptor CHRM3 or CHRNE with or without the MHC class II antigen DRA. To identify the PV antibodies most specific to the disease process, we sorted the data based on the ratio of patient to control frequencies of antigen recognition. The frequency of antigen recognition by patients that exceeded that of control by 10 and more times were the molecules encoded by the CD33, GP1BA, CHRND, SLC36A4, CD1B, CD32, CDH8, CDH9, PMP22 and HLA-E genes as well as mitochondrial proteins encoded by the NDUFS1, CYB5B, SOD2, PDHA1 and FH genes. The highest specificity to PV showed combinations of autoantibodies to the calcium pump encoded by ATP2C1 with C5a receptor plus DSC1 or DSC3 or HLA-DRA. The results identified new targets of pemphigus autoimmunity. Novel autoantibody signatures may help explain individual variations in disease severity and treatment response, and serve as sensitive and specific biomarkers for new diagnostic assays in PV patients.

Background: Previous studies suggested that mitochondrial antibodies contribute to pemphigus vulgaris (PV). Results: PV sera elicited mitochondrial damage, and mitochondria-protecting drugs exhibited protective effect in cell culture and mouse skin. Conclusion: PV antibodies altered O 2 respiration, disrupted electron transfer chain, and increased reactive oxygen species. Significance: Results provide the mechanism of therapeutic action and justify the use of mitochondria-protecting drugs in PV.

c Routine serodiagnosis of herpes simplex virus (HSV) infections is currently performed using recombinant glycoprotein G (gG) antigens from herpes simplex virus 1 (HSV-1) and HSV-2. This is a single-antigen test and has only one diagnostic application. Relatively little is known about HSV antigenicity at the proteome-wide level, and the full potential of mining the antibody repertoire to identify antigens with other useful diagnostic properties and candidate vaccine antigens is yet to be realized. To this end we produced HSV-1 and -2 proteome microarrays in Escherichia coli and probed them against a panel of sera from patients serotyped using commercial gG-1 and gG-2 (gGs for HSV-1 and -2, respectively) enzyme-linked immunosorbent assays. We identified many reactive antigens in both HSV-1 and -2, some of which were type specific (i.e., recognized by HSV-1-or HSV-2-positive donors only) and others of which were nonspecific or cross-reactive (i.e., recognized by both HSV-1-and HSV-2-positive donors). Both membrane and nonmembrane virion proteins were antigenic, although type-specific antigens were enriched for membrane proteins, despite being expressed in E. coli. Herpes simplex virus 1 (HSV-1) and HSV-2 cause significant human morbidity. HSV-2 is the causative agent of most recurrent genital herpes lesions and is sexually transmitted. Infections are often asymptomatic, and most infected individuals are unaware of the infection, yet HSV-2 is associated with an increased risk of HIV acquisition (33) and an increased risk during pregnancy of spontaneous abortion, premature birth, and perinatal herpes (12, 13). Unawareness of HSV-2 infection is also a major contributing factor to transmission to uninfected partners (64,65). In contrast, HSV-1 is usually transmitted during childhood and is found to be associated predominantly with orolabial infections (cold sores). Also, HSV-1 infection of the eye (ocular herpes) is the most common cause of infectious corneal blindness in industrialized countries. Both HSV-1 and HSV-2 establish lifelong latent infections within the dorsal root and trigeminal ganglia and are characterized by periodic reactivation and virus shedding from mucocutaneous epithelium. Owing to the different natural histories and outcomes of HSV-1 and -2 infections, accurate diagnosis of the HSV type is important for patient management and prognosis and controlling potential transmission. For example, knowing the specific HSV type can help the patient take appropriate precautions to prevent transmission of the disease to others. In particular, the identification of unrecognized HSV-2 infection can be used to carefully monitor virus shedding during pregnancy and minimize the risk of perinatal infection.Laboratory tests for HSV infection include virus culture, virus neutralization, PCR, and serological tests. Virus culture is considered the "gold standard" in the early stages of a primary infection. However, it is less sensitive during the healing stage of infection or during recurrent infections. Culture testi...

We performed partial evaluation of pemphigus vulgaris (PV) autoantibody profile using the protein array technology. The sera from seven patients with acute PV and five healthy donors were probed for the presence of autoantibodies characteristic of the organ-non-specific autoimmune disorders rheumatoid arthritis, lupus erythematosus, scleroderma, diabetes and some other autoimmune disorders, but not to desmosomal proteins. The array targeted 785 human genes amplified using Mammalian Gene Clone Collection with gene-specific primers containing 20-bp nucleotide extension complementary to ends of linear pXT7 vector. The array identified PV antibodies significantly (P < 0.05) differentially reactive with 16 antigens, most of which were cell-surface proteins, such as CD2, CD31, CD33, CD36, CD37, CD40, CD54, CD66c and CD84 molecules, nicotinamide ⁄ nicotinic acid mononucleotide adenylyltransferase, immunoglobulin heavy chain constant region gamma 2 and others. Reactivity with Fc-IgG helps explain an ability of the chimeric desmoglein constructs to absorb out all disease-causing PV antibodies. Anti-M 1 muscarinic receptor antibody was also identified, consistent with the facts that while blockade of this receptor causes keratinocyte detachment, its activation is therapeutic in PV. Further proteomics analysis of PV antibodies should help elucidate the immunopathogenic mechanisms underlying keratinocyte detachment and blistering.

Toxoplasma gondii causes congenital toxoplasmosis in newborns resulting with fetal anomalies. Determining the initiation time of infection is very important for pregnant women and current serological assays have drawbacks in distinguishing the recently acute toxoplasmosis. Diagnosis of recently acute infection may be improved by using stage specific antigens in serological assays. In the present study, the diagnostic value of sporozoite specific SporoSAG, bradyzoite specific BAG1 proteins and GRA1 protein expressed by all forms of the parasite have been evaluated ELISA using sera systematically collected from mice administered orally with tissue cyst and oocysts. The anti-SporoSAG IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 1, 10, and 15 (P<0.01). The anti-BAG1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly at days 15, 40, and 120 (P<0.05). The anti-GRA1 IgM antibodies in sera obtained from mice infected with oocysts peaked significantly at days 2, 10, and 40 (P<0.01). The anti-GRA1 IgM antibodies in sera obtained from mice infected with tissue cysts peaked significantly only at day 40 (P<0.05). The anti-SporoSAG, anti-BAG1, and anti-GRA1 IgG titers of mice showed significant increases at day 40 (P<0.05) and decrement started for only anti-GRA1 IgG at day 120. The presence of anti-SporoSAG IgM and IgG antibodies can be interpreted as recently acute infection between days 10–40 because IgM decreases at day 40. Similarly, presence of anti-BAG1 IgM and absence of IgG can be evaluated as a recently acute infection that occurred 40 days before because IgG peaks at day 40. A peak in anti-GRA1 antibody level at first testing and reduction in consecutive sample can be considered as an infection approximately around day 40 or prior. Overall, recombinant SporoSAG, BAG1 and GRA1 proteins can be accepted as valuable diagnostic markers of recently acute toxoplasmosis.

It is well established that auto/paracrine acetylcholine (ACh) is essential for wound epithelialization, and that the mechanisms include regulation of keratinocyte motility and adhesion via nicotinic ACh receptors (nAChRs). Keratinocyte nAChRs can be also activated by non-canonical ligands, such as secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein (SLURP)-1 and -2. In this study, we determined effects of recombinant (r)SLURP-1 and-2 on migration of human epidermal and oral keratinocytes under agarose and epithelialization of cutaneous and mucosal excisional wounds in mice, and also identified nAChRs mediating SLURP signals. Both in vitro and in vivo, rSLURP-1 decreased and SLURP-2 increased epithelialization rate. The mixture of both peptides accelerated epithelialization even further, indicating that their simultaneous signaling renders an additive physiologic response. The specificity of rSLURP actions was illustrated by similar effects on cutaneous and oral wounds, which feature distinct responses to injury, and also by abrogation of rSLURP effects with neutralizing antibodies. rSLURP-1 acted predominantly via the α7 nAChR-coupled upregulation of the sedentary integrins α2 and α3, whereas SLURP-2—through α3, and α9 nAChRs upregulating migratory integrins α5 and αV. The biologic effects of rSLURPs required the presence of endogenous ACh, indicating that auto/paracrine SLURPs provide for a fine tuning of the physiologic regulation of crawling locomotion via the keratinocyte ACh axis. Since nAChRs have been shown to regulate SLURP production, cholinergic regulation of keratinocyte migration appears to be mediated by a reciprocally arranged network. The cholinergic peptides, therefore, may become prototype drugs for the treatment of wounds that fail to heal.

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