Patients
We reviewed the records of vasculitis patients with cutaneous manifestations who underwent skin biopsies in our department between December 2018 and February 2020. All tissue specimens were obtained by skin biopsy, fixed in 10% formalin, step-sectioned and stained with hematoxylin and eosin (HE) and elastica van Gieson staining. A diagnosis of vasculitis in skin tissue specimens required the presence of necrotizing vasculitis, such as fibrinoid degeneration, nuclear dust, neutrophilic infiltration and erythrocyte extravasation in the dermis and subcutaneous fat tissue, and leukocytoclastic vasculitis, such as nuclear dust, neutrophilic infiltration and erythrocyte extravasation in the upper and mid-dermis. The patients were divided into two groups based on skin biopsy findings. Group 1 consisted of eight patients who had histopathological necrotizing and/or leukocytoclastic vasculitis in their skin biopsy specimens. Group 2 consisted of seven patients who were diagnosed as systemic vasculitis but had no evidence of histopathological leukocytoclastic and/or necrotizing vasculitis in their skin biopsy specimens. Ten healthy persons of comparable sex and age distributions were recruited as normal controls. Sera were collected at the same time as the skin biopsies. For sera preparation, blood was centrifuged at 1500 g for 15 minutes. Sera were subsequently aliquoted and stored frozen at −80°C until use.
ELISA for aPT/PT Abs
aPS/PT Ab levels in serum samples were tested using IgG and IgM QUANTA Lite™ aPS/PT (INOVA Diagnostics). Briefly, the serum samples diluted to 1:101, and then added to 96-well plates coated with PS/PT where they were incubated for overnight at 4°C. Add 400 ul of 1:40 diluted horseradish peroxidase (HRP) wash buffer plus to each well then discard all liquid. Repeat this step for a total of three washes. Add 100 ul of polyclonal gout anti-human IgG or IgM Abs labelled with HRP conjugate solutions to each well, and incubate for 1 hour then wash four times. Add 100 ul of 3, 3’, 5, 5’-tetramethylbenzidine chromogen to each well and incubate in the dark for 45 minutes at room temperature. After 100 ul of HRP stop solution addition, measure the absorbance at 450 nm immediately. Each patient serum sample was run in triplicate. Standard calibrators were prepared as the instruction manual.
Rats
Inbred wild-type Wistar rats (4 weeks old) were purchased from Sankyo Laboratory (Sapporo, Japan) and maintained under specific pathogen-free conditions.
Experimental protocols
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Rats (n=4) were given a subcutaneous injection of 0, 2.5, 25, and 250 µg/ml cell-free histones (calf thymus-derived histones containing unfractionated whole histones; Sigma-Aldrich, St. Louis, MO, USA) on the back (300 µl/site). Two hours later, the skin was resected for immunostaining using a rat IgM class aPS/PT monoclonal Ab [10]. Formalin-fixed, paraffin-embedded skin tissues were cut into 4 µm sections, and the sections were allowed to react with 1 µg/ml aPS/PT monoclonal Ab (rat IgM) followed by 2 µg/ml Alexa Fluor 594-conjugated goat anti-rat IgM Ab (Abcam, Cambridge, UK). Thereafter, the sections were mounted using a solution containing 4¢,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA).
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Rats (n=19) were given a subcutaneous injection of 250 µg/ml cell-free histones and then divided into four groups: Group A with an intravenous administration of 1.25 µg/g weight of the rat IgM class aPS/PT monoclonal Ab 2 hours after priming by histones (n=5; histone isc with aPS/PT iv), Group B with rat IgM (eBioscience, San Diego, CA, USA; n=4; histone isc with IgM iv), Group C with rat IgG (eBioscience; n=5; histone isc with IgG iv), and Group D without immunoglobulin administration (n=5; histone isc). One week later, all rats were euthanized for histopathological analyses.
Statistics
Statistical analysis was performed using the Mann-Whitney U test for the comparison of Ab levels, Fisher’s exact probability test for the comparison of frequencies, and Bonferroni’s test for multiple comparisons was also used. A p-value of less than 0.05 was considered to be statistically significant. All data are expressed as mean ± standard deviation.