18 Q^5 t]HKn
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number KqIe8bi^G
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The rO>
wX_
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at eMMiSO!3
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The gGz_t,=
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488
k`zK
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, si4=C
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive nQ*oOxe|X
control for Ca2+ induction. The data thus obtained was analyzed with the software Win H_$f
v_
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on CZno2$8@e
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 @)B5^[4(;
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were |&*rSp2iH
harvested and fractionated as detailed above, and the S-100 fractions were assessed by H0Xda.Y(
immunoblotting for presence or absence of annexin I. t#Yh!L6>
Mouse bone marrow transduction and transplantation. lk'jBl%
Retrovirus-mediated transduction of mouse bone marrow cells was done by published XSIO0ep
methods49. Prestimulated low-density bone marrow cells were infected with high-titer 78A4n C
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated
T[K?A+l
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing )u:8Pv
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by iFT3fP'> 5
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) vYun^(_-
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone ^8t*WphZC
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis 1Ee>pbd
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of [_GR'x'0x
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was $9b6,Y_-
confirmed by immunoblot analysis. :njUaMFoMA
Determination of renal morphology N1+]3kt ~
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an _dY}86{
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, -Euy5Y
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner FiH!)6T
for morphologic alterations, as previously detailed B9 {DO
Patient population z#!Cg*K(
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) r F-yD1
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per ;]e"bX
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) e1a %Rj~
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. 8BdeqgU/_
The Mayo Institutional Review Board and the Research Ethical Board, University Health Y`Rf E
Network, University of Toronto approved the study protocol. All patients gave written jV8><5C
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or auv\fR :
19 ,1kV9_x
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 ZaV@}=Rd8
months were not included in the study. Patients with active infection, diabetes, or a D~,R@7
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), x F
#)T*
medications, malignancies) were also excluded. /otgFQ_
Treatment p*,mwKN:
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of [HfFC3U
high-quality protein) diet was recommended and patients were encouraged to maintain #v<+G=r*O
the same diet throughout the duration of the study. All patients received a similar &0TheY;srf
conservative treatment regimen that included loop diuretics to control edema, an "+60B0>sc
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. )}8%Gs4C
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added +cN2 KP
when required to control systolic blood pressures to <135 mm Hg in >75% of the Q.yoxq
readings. Patients who after a minimum of 4 months of conservative therapy and ];YOP%2
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of WBdb[N6\
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions, [8n4lE[)"
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine #Ba'k6b
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given I1jF`xQ&0
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count u8QX2|
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count <Th.}=
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per <o
O_wS@:
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a ljRR
second course of rituximab treatment following the same protocol described above. a6C~!{'nW
Follow-up <T>f@Dn,
In all patients, clinical and laboratory parameters including complete blood counts, g=wnly
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum -q27N^A0
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry XR(kR{yo
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion %!RQ:?=
in the urine were assessed by performing two consecutive 24-h urine collections at each c3+vtP&
time point. Data were considered accurate when urinary creatinine excretion was !xSGZD=AD
consistent with a complete 24 h collection. The mean of the two measurements was e"Z~%,^A
considered for the analysis. The presence of HACAs was evaluated at baseline and at `B~%TEvMh
months 3, 6, 9, and 12. {^F_b% a4z
Method / Approach / Study/ Technique +9t@eHJT1
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In addition, tissues were stained for infiltrating lymphocytes (CD20 and CD3), and the vg<_U&N=-r
amount of interstitial fibrosis was quantified by histomorphometry. Formalin-fixed, u
VB&DE
paraffin-embedded sections were cut onto coated glass slides. Following heat-induced %
nR:Rc!
antigen retrieval, sections were incubated at 20°C overnight with either anti-CD20 H&I0\upd
primary antibody or anti-CD3 primary antibody, both at 1:1000 dilution (Dako, Canada 6;ICX2Wq'
Inc, Mississauga, Ontario, Canada). After rinsing all sections, pretreatment with 3% Mb-C DPT
hydrogen peroxide was performed to prevent endogenous peroxidase activation. Sections xi ^_C!*J
were incubated with a secondary rabbit anti-mouse antibody linked with avidin–biotin 8YCtU9D
complex. Sections were counterstained with hematoxylin and examined by light wEk9(|
microscopy. n{M!l\1
The HACA assay is a proprietary bridging enzyme-linked immunosorbent assay Oa{M9d,l
performed at Genentech Inc. that measures the antibody response to rituximab in human j5AW}
serum samples. T. {P}#'|
In all patients, clinical and laboratory parameters including complete blood counts, /Z-|E
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum 5'@}8W3b
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry M^H357r%
and at months 1, 3, 6, 9, and 12. j
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