18 |@nvg>mu
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number Pms3X
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The *,
*"G?
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at ?RpT_u
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The )SA$hwR
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 3jS=
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, TP{a*ke^5,
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive XOqpys
control for Ca2+ induction. The data thus obtained was analyzed with the software Win }0 <x4|=
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on {+xUAmd
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 >.{
..~"K
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were eaX`S.!jR
harvested and fractionated as detailed above, and the S-100 fractions were assessed by h*MR5qa
immunoblotting for presence or absence of annexin I. ''5%5(Y.r
Mouse bone marrow transduction and transplantation. ]`.
d%Vx
Retrovirus-mediated transduction of mouse bone marrow cells was done by published m/eGnv;!
methods49. Prestimulated low-density bone marrow cells were infected with high-titer :ZL>JVk
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated EW5S%Y
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing }2NH>qvY
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by SAH\'v0
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) }qK
eX4\-
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone UkqLLzL
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis dlT\VWMha(
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of vj^UF(X
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was N!h>fE`
confirmed by immunoblot analysis. %<M<'jxSca
Determination of renal morphology g$ 2M|Q
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an J:(Shd'4D
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, ]Ea7b
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner *z+\yfOO"
for morphologic alterations, as previously detailed zHdp'J"
Patient population J.nVEqLZ
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) ^&6NB)6
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per (Dl"s`UH~
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) w-#0k.T
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. 7S1!|*/
I
The Mayo Institutional Review Board and the Research Ethical Board, University Health e{}o:r
Network, University of Toronto approved the study protocol. All patients gave written -$0S#/)Z
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or &.z/dFmG
19 RdDcMZ
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 h4qR\LX
months were not included in the study. Patients with active infection, diabetes, or a s{,e^T
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), ~Afs
medications, malignancies) were also excluded. Kulg84<AwM
Treatment ?oJ~3Kg
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of Q:M>!|
high-quality protein) diet was recommended and patients were encouraged to maintain \'EWur"
the same diet throughout the duration of the study. All patients received a similar wMUnZHd{|
conservative treatment regimen that included loop diuretics to control edema, an "_K 6=
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. E+)Go-rS(
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added ;MMFF {
when required to control systolic blood pressures to <135 mm Hg in >75% of the RnrM
rOh
readings. Patients who after a minimum of 4 months of conservative therapy and vjb?N
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of **q/'K
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions, PGKXzp'
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine a82mC r
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given #K
l2K4
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count aOHCr>po,
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count '=H^m D+gl
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per +B m+Pj>
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a f UIs(}US
second course of rituximab treatment following the same protocol described above. SIl
g
Follow-up K_5&_P1
In all patients, clinical and laboratory parameters including complete blood counts, vO_quQ[ .
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum gJ&!w8v.
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry UW*
aSZ/?
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion Q]wM/7
in the urine were assessed by performing two consecutive 24-h urine collections at each _PM<25Y,@
time point. Data were considered accurate when urinary creatinine excretion was Tv$7aVi!
consistent with a complete 24 h collection. The mean of the two measurements was p*0Ve21i,
considered for the analysis. The presence of HACAs was evaluated at baseline and at kYB
<FwwB
months 3, 6, 9, and 12. 0 MIMs#
Method / Approach / Study/ Technique AD\<}/3U
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In addition, tissues were stained for infiltrating lymphocytes (CD20 and CD3), and the 9r nk\`E
amount of interstitial fibrosis was quantified by histomorphometry. Formalin-fixed, "O[76}I+.q
paraffin-embedded sections were cut onto coated glass slides. Following heat-induced Sz|CreFK16
antigen retrieval, sections were incubated at 20°C overnight with either anti-CD20 ,/6:bc:W
primary antibody or anti-CD3 primary antibody, both at 1:1000 dilution (Dako, Canada *ZR@z80i
Inc, Mississauga, Ontario, Canada). After rinsing all sections, pretreatment with 3% `4.sy +2
hydrogen peroxide was performed to prevent endogenous peroxidase activation. Sections g]<Z]R`
were incubated with a secondary rabbit anti-mouse antibody linked with avidin–biotin 'ZH<g8:=@
complex. Sections were counterstained with hematoxylin and examined by light nRB>[lG
microscopy. 8I\eromG
The HACA assay is a proprietary bridging enzyme-linked immunosorbent assay
'e>sHL
performed at Genentech Inc. that measures the antibody response to rituximab in human Ccr+SR2
serum samples. pLQSG}N
In all patients, clinical and laboratory parameters including complete blood counts, Z4AAg
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum |SP.S 0.y
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry NhK(HTsvK
and at months 1, 3, 6, 9, and 12. [k~+(.2I
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