Studies On Association Of Herpes Simplex Virus Type I And Human Herpesvirus 8 With Pemphigus, Bullous Pemphigoid And Psoriasis Vulgaris

Objectives1. To examine human herpesvirus 8 ( HHV - 8) DNA in peripheral mono-nuclear cells ( PBMCs) and IgG antibody in sera from blood donors of Northeast China, and to analyze whether there were differences among the blood donors of different sex, ethnic groups and blood types.2. To examine HHV -8 DNA in skin lesion biopsies, PBMCs and throat swabs, and HHV - 8 specific IgG antibody in sera from patients with pemphigus, bullous pemphigoid and psoriasis vulgaris, to investigate the possible role of HHV - 8 in the pathogenesis of these diseases.3. To examine herpes simplex virus type I (HSV - I ) DNA in skin lesion biopsies, PBMCs, throat swabs, and HSV- I IgM and IgG antibodies in sera from these same patients, in order to analyze the possible association of these diseases with HSV - I.Materials and Methods1. Sample collection(1) Diagnosis confirmationAll the specimens in our studies were obtained from patients with confirmed diagnosis. The diagnosis of psoriasis vulgaris was confirmed by clinical and his-topathological examination. That of pemphigus was confirmed not only by clinical, histopathological examination, but also by direct immunofluorescence (DIF) test, and, in some cases, by indirect immunofluorescence (IIF) test. DIF test on lesional or non - lesional skin showed immunoglobulins and/or C3 deposits in the intracellular space of epidermis, and IIF test showed positive anti - intracellular antibody in patient sera using moused or rat's tongue or esophagusas substrate. The diagnosis of bullous pemphigoid was also confirmed by clinical, histopathologial, and DIF test and/or IIF test. DIF test on lesional or non - lesional skin showed immunoglobulins and/or C3 deposits at dermo - epidermal junction ( DEJ) , and IIF test in which tongue or esophagus of mouse or rat was also used as substrate showed positive anti - basement membrane zone ( BMZ) antibody in patient sera.(2) Blood donors and normal controlsOne hundred and sixty - one samples of peripheral blood from healthy blood donors were collected, from which 161 samples of PBMCs were isolated and 40 samples of sera were separated. Eighteen specimens of normal skin were collect-ed from excised tissue of patients who underwent surgical operation. Twenty normal throat swabs were obtained from healthy subjects.(3) Patients with psoriasis vulgarisTwenty - four skin lesion biopsies, 59 samples of peripheral blood, 48 throat swabs were obtained from 67 patients with psoriasis vulgaris, and then from peripheral blood 43 sera were separated and 59 samples of PBMCs were isolated.(4) Patients with pemphigus and bullous pemphigoidThirty -seven skin lesion biopsies, 16 samples of peripheral blood and 22 sera were collected from 54 patients with pemphigus. Twenty - four skin lesion biopsies, 12 samples of periphery blood and nine sera were obtained from 35 patients with bullous pemphigoid.2. DNA extraction and concentration measurement & adjustment(1) PBMCs were isolated from periphery blood after treatment with red blood cell lysis buffer.(2) DNA was extracted from PBMCs of blood donors, and from skin lesion biopsies, PBMCs and throat swabs from patients with pemphigus, bullous pemphigoid and psoriasis vulgaris with standard phenol - chlorophorm method.(3) DNA concentration and purity were measured with spectrophotometry at 260nm and 280nm, then the concentration was adjusted to 0. 1uLg/ul with TE buffer.3. Primer design(1) A nested primer set GQ1/GQ2 and GQ3/GQ4, designated as HHV -8 DNP primer, was designed to amplify the highly conserved region of HHV -8 DNA polymerase ( DNP) gene fragment.(2) Another nested primer set KS1/KS2 and NS1/NS2, designated as HHV -8 KS330 primer, was designed to amplify the highly specific region of HHV -8 KS330Bam gene fragment.(3) A primer set, designated as PV1245, was designed for detecting the highly conserved region of HSV -I DNA polymerase (DNP) gene fragment.4. PCR amplification(1) Nested PCR with HHV - 8 DNP primer was un...