18 ]8q%bsl+
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number A7I8Z6&
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The A-@-?AR
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at 6w .iEb
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The \a~;8):q=i
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 0z
q\ j
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, e2VL/>y`
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive $D2Ain1
control for Ca2+ induction. The data thus obtained was analyzed with the software Win ;#Crh}~
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on *SAcH_I2$>
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 B=nx8s
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were (t]R#2{
harvested and fractionated as detailed above, and the S-100 fractions were assessed by u0$5Fd&X
immunoblotting for presence or absence of annexin I. a7 '\*
Mouse bone marrow transduction and transplantation. k%bTs+]*
Retrovirus-mediated transduction of mouse bone marrow cells was done by published vr]dRStr
methods49. Prestimulated low-density bone marrow cells were infected with high-titer nG(|7x
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated U]R|ej
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing jak|LOp
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by _ZvX" {
y~
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) 7C6BZ$(
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone =*O9)$b
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis *Sp O|*'
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of kQ:>j.^e
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was 5Qe}v
confirmed by immunoblot analysis. q?b)zeJ
Determination of renal morphology BtDgv.;GH
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an <MDFfnj
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, bgx5{!A
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner >?Y3WPB<F
for morphologic alterations, as previously detailed i_Q4bhVj
Patient population 9H Bx[2&
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) :r[-7
[/
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per 'G
By^hj?
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) m+JG
e5fR<
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. N_~Wu
The Mayo Institutional Review Board and the Research Ethical Board, University Health gj(l&F *@
Network, University of Toronto approved the study protocol. All patients gave written ]4pC\0c
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or {Eb2<;1o{
19 r_2VExk
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 Op)R3qt{
months were not included in the study. Patients with active infection, diabetes, or a GVPEene
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), /t2<OU9
medications, malignancies) were also excluded. qv >(
Treatment {d5ur@G1
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of Vam8NnZ|r
high-quality protein) diet was recommended and patients were encouraged to maintain _ -FQ78C
the same diet throughout the duration of the study. All patients received a similar k-p7Y@`+a
conservative treatment regimen that included loop diuretics to control edema, an K0bmU(Xxp
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. [75e\=wK
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added ;R>42
qYF
when required to control systolic blood pressures to <135 mm Hg in >75% of the #SOj4W
readings. Patients who after a minimum of 4 months of conservative therapy and ^C#bW<T
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of gG|
1$
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions, k.VOS0
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine mr\L q~*c
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given iK23`@&%_
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count ))-M+CA
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count 246!\zf
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per 3^Q U4
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a JHOBg{Wg
second course of rituximab treatment following the same protocol described above. G(,~{N||
Follow-up b-gVRf#F
In all patients, clinical and laboratory parameters including complete blood counts, fu;B ?mIn
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum M/I d\~
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry Uv$u\D+@[
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion FK('E3PG
in the urine were assessed by performing two consecutive 24-h urine collections at each DccsVR`7
time point. Data were considered accurate when urinary creatinine excretion was F#R\Ot,hv
consistent with a complete 24 h collection. The mean of the two measurements was ]@ g$<&
considered for the analysis. The presence of HACAs was evaluated at baseline and at QAw,X Z.K^
months 3, 6, 9, and 12. Tj~#Xc
Method / Approach / Study/ Technique xL"o)]a=
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