Although methotrexate (MTX) is one of the most clinically effective therapies employed to treat psoriasis, the mechanism by which low-dose MTX acts to modulate the hyperplasia of psoriasis, leading to the restoration of clinically normal skin, is only partially understood. MTX has been considered a cytotoxic agent that mediates its effect primarily on proliferating or cycling epidermal cells. Recently, proliferating lymphoid cells have been identified in psoriatic lesions, raising the possibility that proliferating lymphoid cells could be another target cell that is killed by MTX. In this study, we examined the growth-inhibitory and cytotoxic effects of MTX on proliferating lymphoid cells [THP-1 (macrophage), and MOLT-4 (T cell)], epithelial cells (HeLa, and HaCat), and normal human keratinocytes (NHK) in vitro. The proliferating cells were exposed to MTX for 24 h, and placed in fresh media to mimic the transient MTX blood levels that result from once-weekly therapy. THP-1 and MOLT-4 were found to be 10-100 times more sensitive to the cytotoxic effects of MTX than were HeLa and HaCat, and more than 1000 times more sensitive than primary human keratinocytes. At MTX concentrations that would be expected to occur in vivo during once-weekly therapy, a large percentage (> 95%) of proliferating lymphoid targets would be killed, and only a small percentage (< 10%) of proliferating epidermal cells would be affected. This in vitro data suggests that in psoriasis proliferating lymphoid cells are more likely than epithelial cells to be a major cellular target of MTX in vivo.

DNA amplification-restricted transcriptiontranslation (DARTT), is based on DNA amplification by the polymerase chain reaction (PCR) and uses PCR to truncate protein-encoding DNA while adding transcriptional and translational initiation signals to the segment. The amplified DNA segments are transcribed into RNA and translated into protein in vitro and the synthesized proteins are used to define functional sites. DARTT was applied to rhesus rotavirus gene segment 4 cDNA in order to create a series ofcarboxyl-terminal truncations and new amino termini in the encoded VP4 capsid protein.The truncated VP4 polypeptides were tested for reaction with 11 VP4-specific neutralizing monoclonal antibodies to identify the minimum polypeptides required for antibody recognition. Monoclonal antibodies 2G4, M2, and M7, which neutralize a number of serologically distinct rotaviruses, required amino acids 247-474 of VP4 for binding. DARTT is potentially applicable to the identification of discontinuous epitopes and functional domains on a variety of proteins.Rotavirus infection is a leading cause of infant mortality in developing countries and an important cause of acute gastroenteritis in young children in the United States (1-3). Rotaviruses contain 11 double-stranded RNA segments inside a double-capsid icosahedral shell. Natural rotavirus infections result in the production of neutralizing antibodies to the outer capsid proteins, . VP4 is an 86.5-kDa, nonglycosylated protein encoded by gene segment 4 of rhesus rotavirus (RRV) (4). VP4 is cleaved by trypsin to an amino-terminal fragment, VP8 (amino acids 1-246), and a carboxyl-terminal fragment, VP5 (amino acids 247-776), and this cleavage is associated with enhanced viral infectivity (5). Genetic and biochemical studies indicate that VP4 is associated with viral virulence (6, 7) and cell penetration (8). VP4 is responsible for binding the erythrocyte protein glycophorin (9) and is the viral hemagglutinin (10). Antibodies directed at VP4 inhibit viral hemagglutination, neutralize the virus in vitro (11), and passively protect mice against rotavirus challenge in vivo (12). VP4 also effectively induces protective immunity in animals and is immunogenic in young children (13-16).In previous studies of monoclonal antibody (mAb) "escape" mutants, we have identified point mutations in VP4 that appeared to be involved in viral neutralization (4,17). However, this analysis did not determine whether neutralizing antibodies actually bound to these regions or whether the identified mutations elicited conformational changes at a distant site. In order to directly define the minimal VP4 polypeptides that are bound by selected neutralizing antibodies, a broadly applicable method for synthesizing a series oftruncated polypeptides has been devised. The procedure is called DNA amplification-restricted transcription-translation (DARTT) and is based on the polymerase chain reaction (PCR) (18)(19)(20). We have applied DARTT analysis to the coding region of gene segment 4 of RRV and determined ...

A 27‐year‐old white man with a tattoo of 3 months’ duration was referred for possible laser removal because a pruritic eruption had developed in the tattoo 1 month after placement. Physical examination revealed erythematous papules confined to the red areas of the tattoo ( Fig. 1). He also had flat‐topped papules on the glans penis, which the patient stated had developed a few weeks after tattoo placement. 1 Tattoo before treatment, showing involvement of only the red areas Biopsy of a penile lesion revealed a histologic pattern characteristic of lichen planus. Biopsy of the tattoo eruption revealed a lichenoid lymphocytic infiltrate with hyperkeratosis, hypergranulosis, and jagged epidermal hyperplasia. Deposits of black granular material were also present in the upper part of the dermis ( Fig. 2). 2 Histopathology of the tattoo eruption shows a lichenoid lymphocytic infiltrate with hyperkeratosis, hypergranulosis, and jagged epidermal hyperplasia, with deposits of black granular material in the upper dermis During a 2‐week follow‐up visit, the patient developed more lesions on his arms and legs. After 4 weeks of treatment with halobetasol propionate ointment 0.05% to the tattoo, legs, and arms, all lesions had resolved. The red areas of the tattoo became flat, nonpalpable, and asymptomatic ( Fig. 3). 3 Tattoo after treatment with topical corticosteroids

Given the important theoretical advantages of enzyme-linked immunosorbent assay-based XM methods over the CDC XM, however, further testing of the clinical relevance of the Cross-Stat is warranted.