18 U.wgae].O;
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number \f{C2d/6j
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The bcj7.rh]'h
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at YToRG7X#
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The WgxlQXi-B
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 H%])>
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, <cepRjDn
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive ld2\/9+n
control for Ca2+ induction. The data thus obtained was analyzed with the software Win ,VHvQU
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on KgKV(q=
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 Iqo4INGIi
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were C{Npipd}v
harvested and fractionated as detailed above, and the S-100 fractions were assessed by (6JD<pBm
immunoblotting for presence or absence of annexin I. [_H9l)
Mouse bone marrow transduction and transplantation. TXy*- <#vR
Retrovirus-mediated transduction of mouse bone marrow cells was done by published JQbI^ef_;
methods49. Prestimulated low-density bone marrow cells were infected with high-titer v3aiX
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated
1S_KX.
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing #Q|$&b
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by fT'A{&h|U
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) /nC"'d(#
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone y8,es$
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis ^{Mx?]z
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of
J/
rQ42d
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was B;rq{ac!P]
confirmed by immunoblot analysis. {O!fV<Vx 9
Determination of renal morphology wV(_=LF
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an U;{VL!
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, 4V[+6EV
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner ]Q)TqwYF
for morphologic alterations, as previously detailed =8<SKY&\X
Patient population |pJ.73
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) Lqz}h-Ei
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per c.d*DM}W
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) Ak4iG2
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. {zg}KiNDZd
The Mayo Institutional Review Board and the Research Ethical Board, University Health
beO*|
Network, University of Toronto approved the study protocol. All patients gave written !q$IB?8
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or dyu~T{
19 Aja'`Mu
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 b/<n:*$
months were not included in the study. Patients with active infection, diabetes, or a Ak|jJ
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), VE{t]>*-u
medications, malignancies) were also excluded. *y.KD4@{
Treatment CQ13fu+|6
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of 6B|IbQ^
high-quality protein) diet was recommended and patients were encouraged to maintain MbjH\XRB
the same diet throughout the duration of the study. All patients received a similar "z7.i{
conservative treatment regimen that included loop diuretics to control edema, an ?1 ?m4i
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. pGUrYik4
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added Tpkm\_
when required to control systolic blood pressures to <135 mm Hg in >75% of the P>jlFm
readings. Patients who after a minimum of 4 months of conservative therapy and Pm
V:J9
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of d94Lc-kq^
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions,
,9
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine m<