18 o~(/Twxam
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number BwpSw\\?@
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The &azy1.i~
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at
DC5^k[m
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The T=kR!Gx
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 8}^R jMgI
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, p1blPBlp
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive bS!4vc1`2
control for Ca2+ induction. The data thus obtained was analyzed with the software Win [@PD[-2QG3
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on RdirEH*H
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 0?{Y6:d+
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were VsrY
U@V
harvested and fractionated as detailed above, and the S-100 fractions were assessed by K4{1}bU{>
immunoblotting for presence or absence of annexin I. Si:
$zGL$(
Mouse bone marrow transduction and transplantation. FD+y?U
F
Retrovirus-mediated transduction of mouse bone marrow cells was done by published I_k!'zR[N
methods49. Prestimulated low-density bone marrow cells were infected with high-titer a
St
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated M(8Mj[>>Rj
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing gI
/#7Cr
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by
dYn<L/#
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) L
(#DVF
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone e0nr dM[i
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis dP7nR1GS
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of @N% /v*
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was 0&@pD`K e
confirmed by immunoblot analysis. ylo/]pVs
Determination of renal morphology 6M/*]jLq4
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an 0YApaL+jt
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, Ol@ssm
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner bFv,.(h'
for morphologic alterations, as previously detailed ;G=:>m~
Patient population v1m'p:7uGB
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) @'ln)RT,
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per %_>+K;<
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) ?L'4*S]
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. |vI1C5e
The Mayo Institutional Review Board and the Research Ethical Board, University Health waYH_)Zx
Network, University of Toronto approved the study protocol. All patients gave written D|lm,
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or $zR[2{bg
19 K~MTbdg
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 y=H\Z/=
months were not included in the study. Patients with active infection, diabetes, or a fho$:S
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), i]L4kh5
medications, malignancies) were also excluded. >e4w8Svcy
Treatment ENEn Hu^
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of {TWgR2?{C
high-quality protein) diet was recommended and patients were encouraged to maintain 82q_"y>6
the same diet throughout the duration of the study. All patients received a similar [HV9KAoA
conservative treatment regimen that included loop diuretics to control edema, an EXrOP]Kl
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. potb6jc?
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added ;44?`[oP
when required to control systolic blood pressures to <135 mm Hg in >75% of the [p%OIqC`pB
readings. Patients who after a minimum of 4 months of conservative therapy and FQ72VY
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of pR
VL}^Rk
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions,
@rh1W$
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine 2qY+-yOEt
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given 0aF&5Lk`y
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count ,iUYsY
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count ZV=)`E`I|
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per
r$7D;>*O{
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a sT^^#$ub
second course of rituximab treatment following the same protocol described above. A^a9,T
Follow-up HKr6h?Si^
In all patients, clinical and laboratory parameters including complete blood counts, 9bDxml1
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum -/s2'
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry cX64 X
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion A#"AqNVWv
in the urine were assessed by performing two consecutive 24-h urine collections at each d9hJEu!Lu
time point. Data were considered accurate when urinary creatinine excretion was x{B%TM-Ey
consistent with a complete 24 h collection. The mean of the two measurements was O*<,lq 0K
considered for the analysis. The presence of HACAs was evaluated at baseline and at D/jS4'$vA
months 3, 6, 9, and 12. k($N_XlE
Method / Approach / Study/ Technique =a_B' ^`L
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To improve the efficiency of the method, the following approach may be applied. R ks3L
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accepted Vm1U00lM{
20 nl 7=Nhh
This approach will be explained and discussed thoroughly in the body of the report. <tEN1i
This can be accomplished by F:8cd^d~u
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The above preliminary analysis has provided important information zl:
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In addition, tissues were stained for infiltrating lymphocytes (CD20 and CD3), and the FuNc#n>
amount of interstitial fibrosis was quantified by histomorphometry. Formalin-fixed, -Fodqq@,
paraffin-embedded sections were cut onto coated glass slides. Following heat-induced b7It8
antigen retrieval, sections were incubated at 20°C overnight with either anti-CD20 BzqM$F(
L,
primary antibody or anti-CD3 primary antibody, both at 1:1000 dilution (Dako, Canada 6xs_@Vk|d
Inc, Mississauga, Ontario, Canada). After rinsing all sections, pretreatment with 3% JkazB1h
hydrogen peroxide was performed to prevent endogenous peroxidase activation. Sections -.|V S|y
were incubated with a secondary rabbit anti-mouse antibody linked with avidin–biotin ic:_v?k
complex. Sections were counterstained with hematoxylin and examined by light .17WF\1HC.
microscopy. 5MH\Gqe7
The HACA assay is a proprietary bridging enzyme-linked immunosorbent assay 0x Vue[ep
performed at Genentech Inc. that measures the antibody response to rituximab in human 2*6b{}yJH
serum samples. MfhJb_q`
In all patients, clinical and laboratory parameters including complete blood counts, W'2|hP
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum )8
ejT6r
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry GZ#6}/;b
and at months 1, 3, 6, 9, and 12. 'W5r(M4U
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