18 >
5CK&6
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number 3+ 6Ed;P
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The `MU~N_
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at OXEEpoU?V
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The
]=~dyi
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 >of34C"DI
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, \l59/ZFan
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive g6,D Bkv2
control for Ca2+ induction. The data thus obtained was analyzed with the software Win /.SG? 5t4
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on j.q}OK
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 ,P a*; o\
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were `==l2AX
harvested and fractionated as detailed above, and the S-100 fractions were assessed by y
\skke]
immunoblotting for presence or absence of annexin I. QL7b<xDQC*
Mouse bone marrow transduction and transplantation. zy,SL
|6:
Retrovirus-mediated transduction of mouse bone marrow cells was done by published 3}|[<^$
methods49. Prestimulated low-density bone marrow cells were infected with high-titer K0gQr.J53
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated 4LTm&+(5
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing p
{w-
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by Fl`U{03
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) r9ulTv
}X
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone H,(vTthd
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis k6bct@7
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of OFtaOjsyUa
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was CTNL->
confirmed by immunoblot analysis. t{>K).'
Determination of renal morphology e8vy29\S
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an 6|eqQ+(A
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, +YvF+E
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner On*I.~
for morphologic alterations, as previously detailed "-&K!Vfs
Patient population a
8k2*u
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) :BPgDLL,
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per `H! (hMMV
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) j=O+U_w
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. sBadiDG~9
The Mayo Institutional Review Board and the Research Ethical Board, University Health 8Yq06o38C
Network, University of Toronto approved the study protocol. All patients gave written Lp&nO
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or 1K9.3n
19 jF?0,g
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 0S5xmEzop
months were not included in the study. Patients with active infection, diabetes, or a JQKXbsXS
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), 2'fd4rE5
medications, malignancies) were also excluded. dUv@u!}B
Treatment X;!*D
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of ?2"g*Bak
high-quality protein) diet was recommended and patients were encouraged to maintain ;T^s&/>E
the same diet throughout the duration of the study. All patients received a similar 9?\cm}^?
conservative treatment regimen that included loop diuretics to control edema, an ('7?"npd
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. 8[@,i|kgg0
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added ->I.D?p
when required to control systolic blood pressures to <135 mm Hg in >75% of the X4o#kW
readings. Patients who after a minimum of 4 months of conservative therapy and YW7w>}aW
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of ~m fG
Yk"
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions,
cpk\;1&t
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine 7Q!ksp
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given ,It0brF
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count QI[WXxp
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count le^Fik
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per P 'h39XoZ
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a :NLY;B`
second course of rituximab treatment following the same protocol described above. e^*&&
Follow-up EF&CV{Sw
In all patients, clinical and laboratory parameters including complete blood counts, GpPM ?
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum n_{&dVE
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry ;jxX /c
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion d %}?%VH
in the urine were assessed by performing two consecutive 24-h urine collections at each y={ k7
time point. Data were considered accurate when urinary creatinine excretion was :[l}Bb,
consistent with a complete 24 h collection. The mean of the two measurements was eLT3b6'"?
considered for the analysis. The presence of HACAs was evaluated at baseline and at t_ CMsp
months 3, 6, 9, and 12. ();Z,A
Method / Approach / Study/ Technique )T '?"guh`
A discussion is presented of a problem-solving system MZVbOcSAd
To improve the efficiency of the method, the following approach may be applied. Kez0Bka
In order to an investigation was made to find the causes of the 7/&t