healthmedicinet

The Hadassah Medical Center Institutional Review Board (IRB) approval was obtained
for this study (IRB protocol number and version: EFA-EFE-IV-01), and all of the study
procedures were carried out in accordance with the IRB guidelines. This study followed
the tenets of the Helsinki Declaration.

Human Corneal Epithelial (HCE) cells and Human Conjunctival Fibroblasts (HCF)

HCE cells were obtained from human corneoscleral rim explants, taken from three different
human donors, provided by the Department of Ophthalmology at the Hadassah Medical
Center, using a previously described method 26]. HCE cells were cultured in supplemented hormonal epithelial medium (SHEM) 27]. HCE cells were incubated at 37 °C under 95 % humidity and 5 % CO
₂
. The culture medium was replaced every other day. Cultures were kept for 10 to 14 days
until a density of 90 % confluence was observed. At this time, cells were passaged
and seeded onto 6-well plates at a density of 2.0 x10
⁵
cells/well. Cells were observed by phase-contrast microscopy to ensure uniformity
of morphology. The purity of HCE cultures was confirmed by staining for cytokeratin-
19with the indirect immunoperoxidase procedure with a monoclonal antibody to human
cytokeratin-19 (Abcam, UK). Second generation cells were used in all experiments.

Human conjunctiva explant cultures were established using specimens obtained at the
time of strabismus surgery. The human conjunctival explants were taken from four different
human donors. The isolation and culture of cell explants were performed within 1–3
h after the strabismus surgery. HCF cells were cultured as previously described 28]. In brief, HCF cells were cultured in fibroblast medium, which contained Dulbecco’s
modified Eagle medium (DMEM) with nutrient mixture F12 (Gibco), supplemented with
4 mM glutamine, 10 % fetal calf serum (FCS), 100 U of penicillin, and 100 ?g of streptomycin/ml.
HCF were incubated at 37 °C under 95 % humidity and 5 % CO
₂
. The medium was replaced every 2–3 days. Cultures were kept for 10 to 14 days until
a density of 90 % confluence was observed.

Culture of human peripheral blood mononuclear cells (PBMCs)

Human PBMCs were isolated from buffy coats provided by the blood bank of the Hadassah
Medical Center. The blood donors were four healthy males aged between 25 and 45. PBMCs
were isolated as previously described 29], 30]. Briefly, whole blood was transferred via tubes covered with anticoagulant and mixed
with RPMI in a 1:1 ratio. Ficoll was added to a new tube in a 1:1 ratio to the whole
blood (originally before the blood/RPMI mixture), with an additional 3 mL of Ficoll.
Blood/RPMI mixture was added to the Ficoll in a slow stream to maintain the gradient,
and the tube was centrifuged for 30 min at 1600 rpm. The top layer (plasma) was aspirated
and the buffy coat (the interface) was removed with a tube and mixed with RPMI to
comprise a 50 ml total mixture. The top layer was aspirated and the cells were covered
once again with new RPMI (50 ml) and centrifuged for another 10 min. The top layer
was aspirated, and 1.0 ml of monocyte isolation buffer was added.

Fatty acids

Alpha linolenic acid (ALA; 18:3 n-3) was obtained as 99 % pure sodium salt (Nuchek
Prep Inc, Elysian, Minnesota, USA). ALA was dissolved in distilled water in a nitrogen
chamber, filtered through a 0.2-?m-pore-size filter, divided into aliquots, and sealed
under nitrogen in opaque eppendorf tubes. ALA was stored at ?80 °C for no longer than
90 days before use. Fatty acids were conjugated with bovine serum albumin (BSA, Fraction
V, Mercury, Israel) at a 5:1 molar ratio before treatment. In order to avoid oxidative
effects, the fatty acids were defrosted once and were not reused again.

Experimental designs

HCF and HCE cells were seeded into 6 wells plates for 72 h (density of 1.2×10
⁵
cells/well) in 2.0 ml of medium. To induce inflammation, HCF and HCE cells were treated
with different mixtures of pro-inflammatory cytokines and Lipopolysaccharide (1 to
100 ng/ml) combinations (Table 1). Culture medium was sampled at 24, 48 and 72 h, and nitrite accumulation was measured
at each time period. HCF and HCE cells treated with combinations 1 to 5 (Table 1) were lysed at 72 h to extract RNA for RT-PCR analysis.

Table 1. The pro-inflammatory combinations added to cultures of HCE cells and HCF

Production of nitric oxide may promote inflammation in mononuclear cells as previously
described 31]. Our preliminary results showed that PBMC released high amounts of nitrite after
treatment with LPS only. Co-cultured human PBMCs (density of 2.0 x 10
⁵
cells/well) with HCF in 2.0 ml of medium were treated with LPS only (1 to 1000 ng/ml
LPS). In addition, co-cultured PBMC with HCF were stimulated by LPS at similar conditions
to those of PBMC and HCF alone. Culture medium was sampled at 24, 48 and 72 h. Nitrite
accumulation was measured at each time period.

HCF, HCE cells, and human PBMCs were pre-incubated for two hours with 200 ?M ALA before
treatment with the inflammatory stimulus 32].

Griess reaction

NO is unstable in culture medium. Nitrite accumulation, an indicator of NO synthesis,
was measured in the culture medium by Griess reaction at 24, 48 and 72 h 33]. Briefly, 80 ?L of cell culture medium was mixed with 20 ?L of Griess reagent [equal
volumes of 1 % (w/v) sulfanilamide in 5 % (v/v) phosphoric acid and 0.1 % (w/v) naphthylethylenediamine-HCl]
and incubated at room temperature for 10 min. The absorbance at 550 nm was then measured
using a microplate reader. Fresh culture medium was used as a blank in all experiments.
The amount of nitrite in the test samples was calculated from a sodium nitrite standard
curve.

RNA isolation

Following treatment, total RNA was extracted from the HCF and HCE cell samples with
RNAqueous Kit (Ambion, USA) following the manufacturer’s instructions. Quantification
of total RNA was performed in a NanoDrop spectrophotometer (ND-1000, USA). RNAs were
stored at ?80 °C until further utilization.

cDNA synthesis

cDNA was synthesized from purified and concentrated 0.5 ?g RNA from each sample using
a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ABI, USA). A 20 ?l
total reaction volume was made with 10 ?l RNA, 2 ?l 10X RT buffer, 0.8 ?l dNTP Mix
(100 mM), 2.0 ?l 10X RT random hexamer primers, 1.0 ?l MultiScribe™ reverse transcriptase,
1 ?l RNase inhibitor and 3.2 ?l nuclease-free water. Synthesis was carried out in
an ABI 7900 Thermo cycler (ABI, USA) and RT-PCR reaction conditions were comprised
of: 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 mins. cDNA samples were stored
at ?20 °C until use.

Real-time polymerase chain reaction

Real-time polymerase chain reaction (Real-time PCR) was performed using TaqMan ® Gene
Expression Assays (ABI, USA) in the ABI Prism 7900HT Sequence Detection System (ABI,
USA) as previously described 34], 35]. Negative controls were included to evaluate DNA contamination of isolated RNA and
reagents.

Real-time PCR assays for iNOS probe (ABI, USA) were performed. An amount of 1 ?L cDNA
was loaded in each reaction, to a total volume of 20 ?l of reaction mixture, and assays
were performed in triplicates.

The fold change in gene expression was normalized to the expression of the endogenous
gene hypoxanthine phosphoribosyltransferase 1 (HPRT1; ABI, USA). Quantitative analysis
was performed using the comparative (??C
_T
) method 34], 35] The results were analyzed by DataAssistâ„¢ SoftwareV 2.0 (ABI, USA).

Statistical analysis

All tests were carried out on three independent cell cultures, and performed in triplicate
for each of the treatments. In the experiments of the effect of ALA on nitrite secretion
in HCF and effect of ALA on NOS-2 mRNA expression tests, we carried out the tests
on four independent cell cultures, and performed triplicates for each of the treatments.

Statistical analysis and multiple comparisons were performed by one-way ANOVA using
the InStat software version 3.0 (InStat software Inc., Graphpad, San Diego, CA). The
differences in mean values among treatment groups were determined by ANOVA adjusted
for multiple comparison by the Tukey test, ANOVA adjusted for multiple comparison
by the Student-Newman-Keuls test, and ANOVA with multiple comparison by the Bonferroni
t-test.