18 L~WC9xguDl
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number 9XH}/FcP_O
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The hC nqe
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at 8[z<gxP`?
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The 2I8RO\zR
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 (_9cL,v
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, /5PV|o
nO
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive ^GyGh{@,f
control for Ca2+ induction. The data thus obtained was analyzed with the software Win AUan^Om
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on cAL*Md8+
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 uoryxKRjc~
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were :~%
zX*
harvested and fractionated as detailed above, and the S-100 fractions were assessed by "#3p=}]
immunoblotting for presence or absence of annexin I. U&(TqRi,
Mouse bone marrow transduction and transplantation. 'QMvj` -
Retrovirus-mediated transduction of mouse bone marrow cells was done by published ;&A%"8o
methods49. Prestimulated low-density bone marrow cells were infected with high-titer P ]prrKZe,
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated AXQG
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing G;'=#c
^
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by P+hp'YK1
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) 0Xke26ga
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone -t5DcEAb$
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis DNh{J^S"}w
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of ](z?zDk
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was FTA[O.tiG
confirmed by immunoblot analysis. :k
aHvf
Determination of renal morphology >o#^)LN
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an fG^#G/n2
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, s-z*Lq*
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner .QaHE`e{
for morphologic alterations, as previously detailed HoFFce7
o
Patient population (t{m(;/
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) l$:.bwXXO
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per R>3a?.X
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) 8:iu 8c$
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. ]Q%|69H}B
The Mayo Institutional Review Board and the Research Ethical Board, University Health ~I+}u]J
Network, University of Toronto approved the study protocol. All patients gave written 0(
s
io\
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or TDMyZ!d
19 cITF=
Ez
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 S!/N
lSr<
months were not included in the study. Patients with active infection, diabetes, or a $?dAO}f3O)
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), CQLh;W`Dc
medications, malignancies) were also excluded. lF=l|.c
Treatment ]{[8$|Mg
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of Ww
}qK|D
high-quality protein) diet was recommended and patients were encouraged to maintain {O"?_6',
the same diet throughout the duration of the study. All patients received a similar zeXMi:X
conservative treatment regimen that included loop diuretics to control edema, an 8@S5P$b};
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. 2WA =U]
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added bw
o{
Lw~
when required to control systolic blood pressures to <135 mm Hg in >75% of the dXe.
5XC
readings. Patients who after a minimum of 4 months of conservative therapy and )LA^j|Y}
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of L?@TF;
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions, e<wRA["
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine bi fi02
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given -*?Y4}mK
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count K|H&x"t
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count zEQ<Q\"1
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per {(DD~~)D
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a O1o.^i$-M
second course of rituximab treatment following the same protocol described above. Kb_R "b3v
Follow-up 0I.9m[<Fc
In all patients, clinical and laboratory parameters including complete blood counts, W~1/vJ.*l
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum L"i
B'=
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry ~YIGOL"?
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion =5P_xQx
in the urine were assessed by performing two consecutive 24-h urine collections at each XA[GF6W,Y
time point. Data were considered accurate when urinary creatinine excretion was w"CcWng1
consistent with a complete 24 h collection. The mean of the two measurements was ~&/|J)}
considered for the analysis. The presence of HACAs was evaluated at baseline and at Jmb [d\ /D
months 3, 6, 9, and 12. Z=O 2tR
Method / Approach / Study/ Technique Yx_[v
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accepted 8#%p[TLj
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In addition, tissues were stained for infiltrating lymphocytes (CD20 and CD3), and the Fzk
amount of interstitial fibrosis was quantified by histomorphometry. Formalin-fixed, ~UjFL~K}
paraffin-embedded sections were cut onto coated glass slides. Following heat-induced T0
W B
antigen retrieval, sections were incubated at 20°C overnight with either anti-CD20 g$~3 @zD
primary antibody or anti-CD3 primary antibody, both at 1:1000 dilution (Dako, Canada 65VnH=
Inc, Mississauga, Ontario, Canada). After rinsing all sections, pretreatment with 3% . m_y5J
hydrogen peroxide was performed to prevent endogenous peroxidase activation. Sections ]RmQ*F
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were incubated with a secondary rabbit anti-mouse antibody linked with avidin–biotin o#{D;'
complex. Sections were counterstained with hematoxylin and examined by light 9+.0ZP?
microscopy. -zg,pK$+
The HACA assay is a proprietary bridging enzyme-linked immunosorbent assay
o)KF+[^
performed at Genentech Inc. that measures the antibody response to rituximab in human 3%m2$\
serum samples. p5^,3&
In all patients, clinical and laboratory parameters including complete blood counts, 9^J8V]X
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum M#on-[
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry cu|gM[
and at months 1, 3, 6, 9, and 12. %'j)~
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