Blood Sedimentation

Primary swine macrophage cell cultures were prepared from defibrinated swine blood as previously described by Zsak et al.18 (link). Briefly, heparin-treated swine blood was incubated at 37 °C for 1 hour to allow sedimentation of the erythrocyte fraction. Mononuclear leukocytes were separated by flotation over a Ficoll-Paque (Pharmacia, Piscataway, N.J.) density gradient (specific gravity, 1.079). The monocyte/macrophage cell fraction was cultured in plastic Primaria (Falcon; Becton Dickinson Labware, Franklin Lakes, N.J.) tissue culture flasks containing macrophage media, composed of RPMI 1640 Medium (Life Technologies, Grand Island, NY) with 30% L929 supernatant and 20% fetal bovine serum (HI-FBS, Thermo Scientific, Waltham, MA) for 48 hours at 37 °C under 5% CO₂. Adherent cells were detached from the plastic by using 10 mM EDTA in phosphate buffered saline (PBS) and were then reseeded into Primaria T25, 6- or 96-well dishes at a density of 5x10⁶ cells per ml for use in assays 24 hours later.
ASFV Georgia (ASFV-G) was a field isolate kindly provided by Dr. Nino Vepkhvadze, from the Laboratory of the Ministry of Agriculture1 in Tbilisi, Republic of Georgia9 (link).
Comparative growth curves between ASFV-G, ASFV-GΔMGF13/14-EGFP were performed in primary swine macrophage cell cultures. Preformed monolayers were prepared in 24-well plates and infected at a MOI of 0.1 (based on HAD₅₀ previously determined in primary swine macrophage cell cultures). After 1 hour of adsorption at 37 °C under 5% CO₂ the inoculums were removed and the cells were rinsed two times with PBS. The monolayers were then rinsed with macrophage media and incubated for 2, 24, 48, 72 and 96 hours at 37 °C under 5% CO₂. At appropriate times post-infection, the cells were frozen at ≤−70 °C and the thawed lysates were used to determine titers by HAD₅₀/ml in primary swine macrophage cell cultures. All samples were run simultaneously to avoid inter-assay variability. For immunofluorescence studies, 2 × 10⁶ cells were seeded in 24 well plates on glass coverslips, after the indicated time, coverslips were fixed with 4% paraformaldehyde for 15 min, and washed twice in PBS.
Virus titration was performed on primary swine macrophage cell cultures in 96-well plates. Virus dilutions and cultures were performed using macrophage medium. Presence of virus was assessed either by HA or by fluorescent microscopy three days after being plated, virus titers were calculated by the Reed and Muench method21 and expressed as HAD₅₀ or TCID₅₀ as detected by the presence HA or fluorescence.

The following clinical tests and ocular investigations were performed for all participants: (1) immunological data as, anti-Scl-70 antibody, anti-SS-A anti-body, anti-SS-B anti-body, and ANA antibody were measured by indirect immunofluorescence assay; (2) evaluation of the patient's inflammatory status via C-reactive protein (CRP) level and erythrocyte sedimentation ratio (ESR) analyses; (3) assessment of the patient's mental state via HADS score; (4) OCTA; (5) ocular measurements, include IOP (Goldmann tonometry), VA (Snellen chart), spherical equivalent refractive error, the tear breakup time (BUT), Schirmer's test, tear meniscus height (TMH), and ocular staining score (OSS).
The protocol of BUT, Schirmer's test, TMH, and OSS were examined as we have previously described [16 (link)].
BUT: fluorescein sodium was applied evenly on the ocular surface, and the first tear point film rupture was observed under cobalt blue light, and the time for this to occur was recorded. Less than 10 s was considered positive.
Schirmer's test: after disinfecting the conjunctival sac, one end of a piece of filter paper measuring (5^∗35 mm was folded into a right angle and inserted into the conjunctival sac. Length of the wet region of the paper after 5 minutes was observed, and < 5 mm was considered positive.
TMH: this was measured under infrared light after a blink using Keratograph 5M software.
OSS: a complete evaluation was performed using corneal fluorescein staining in conjunction with conjunctival lissamine green staining. The cornea, the nasal conjunctiva, and the temporal conjunctiva were the three areas of the ocular surface that were taken into consideration for each eye. A score was given to the nasal and temporal conjunctiva based on the amount of spotty conjunctivitis in the palpebral fissure. A positive OSS index was that the score higher than or equal 3.

Based on the electronic medical records of individuals enrolled in this study, the following clinical and laboratory data were reviewed: (1) general demographic data: age at admission, sex; (2) clinical manifestations: length of fever duration before admission, length of illness at primary IVIG treatment, incidence of splenomegaly, incomplete KD, IVIG-resistance KD, coronary artery lesions, and mortality; (3) laboratory indicators: white blood cell count (WBC), neutrophils count, neutrophil-to-lymphocyte count ratio (NLR), platelet (PLT), hemoglobin (Hb), hypersensitive C-reactive protein (Hs-CRP), erythrocyte sedimentation (ESR), PCT, lactic dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), serum electrolytes, serum ferritin, coagulation function, serum lipids, inflammatory cytokines, and other tests. The assessment of laboratory data was collected based on the worst value indicators during the acute period of KD and before the first dose of IVIG therapy. For the individuals of KD-MAS, the laboratory indicators 36–72 h after the first dose of IVIG treatment and at the time of KD-MAS diagnosis were collected for further analysis.