18 QjF.U8
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number URX>(Y}g9^
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The s26:(J
[{
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at h^?[:XBeav
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The ) dwPD
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 6hAeLlU1
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, Wi
hQ
j
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive $zy
Y"yWRZ
control for Ca2+ induction. The data thus obtained was analyzed with the software Win s|%mGt &L
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on Y-gj
X$qGo
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 CPFd 33
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were EX=+TOkAf
harvested and fractionated as detailed above, and the S-100 fractions were assessed by N[){yaj
immunoblotting for presence or absence of annexin I. v3Te+oLg
Mouse bone marrow transduction and transplantation. @X\Sh>H
Retrovirus-mediated transduction of mouse bone marrow cells was done by published ?dC[VYC\^
methods49. Prestimulated low-density bone marrow cells were infected with high-titer CZxQ
z
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated +\\*Iy'xK
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing -r/# 20Y
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by -TjYQ
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) $ 6Nm`[V
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone !|}J{
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis =}Bq"m
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of #HJ F==
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was f?2Y np=@
confirmed by immunoblot analysis. D3HE~zkI
Determination of renal morphology D7 [n^WtL
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an /-8v]nRB
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, n-}:D<\7
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner >XSe[K
for morphologic alterations, as previously detailed 3wX{U8mrg
Patient population ^ B=x-G.
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) A0V"5syY
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per PxfeU2^{0
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) l(v$+
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. h$f/NSct2
The Mayo Institutional Review Board and the Research Ethical Board, University Health uZ]B ?Z%y#
Network, University of Toronto approved the study protocol. All patients gave written ,au64sH
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or kZU8s'C
19 79s6U^vv"
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 ~XsS00TL`G
months were not included in the study. Patients with active infection, diabetes, or a [9BlP
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), cQ6[o"j.
medications, malignancies) were also excluded. ]1)@.b;QR
Treatment }sPY+ZjV
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of >h[tHM
O
high-quality protein) diet was recommended and patients were encouraged to maintain K[~Wj8W0
the same diet throughout the duration of the study. All patients received a similar nI2}E
conservative treatment regimen that included loop diuretics to control edema, an l>~:lBO
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. iL|*g3`-f
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added _q6+]
when required to control systolic blood pressures to <135 mm Hg in >75% of the *uA?}XEfi
readings. Patients who after a minimum of 4 months of conservative therapy and II>X6
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of "Y(S G
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions, ;2L=WR%
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine \RQ5$!O
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given /
s H*if
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count _&
KaI }O
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count h@{@OAu?
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per JYv&I t
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a X@~sIUXx9
second course of rituximab treatment following the same protocol described above. De>pIN;B>
Follow-up e% 5!
In all patients, clinical and laboratory parameters including complete blood counts, & RR
Ora
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum ;A-Ef
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry *,'"\n
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion "H G:by
in the urine were assessed by performing two consecutive 24-h urine collections at each c$e~O-OVD?
time point. Data were considered accurate when urinary creatinine excretion was 3J(STIxg
consistent with a complete 24 h collection. The mean of the two measurements was 9 z3Iwl
considered for the analysis. The presence of HACAs was evaluated at baseline and at (>;~((2
months 3, 6, 9, and 12. *-P@|eg
Method / Approach / Study/ Technique G 8NSBaZe
A discussion is presented of a problem-solving system [fW:%!Y'
To improve the efficiency of the method, the following approach may be applied. =%]dk=n?TN
In order to an investigation was made to find the causes of the ]h|GaHiE
Although large collections of rules and equations have been complied, none are generally L1D%vu`
accepted D!@c,H
20 :^ cA\2=
This approach will be explained and discussed thoroughly in the body of the report. zF{z_c#3@
This can be accomplished by -AffKo
This algorithm to compute the total cost can be described step by step as follows: ^$T>3@rDB
The above preliminary analysis has provided important information 1$
l3-x
Various methods have been proposed for selecting an optimum... XS1>ti|<
These concepts have been applied to +q=/}|
On the basis of the concept mentioned above, N:d" {k
This can be achieved by A46y?"]/30
In addition, tissues were stained for infiltrating lymphocytes (CD20 and CD3), and the Uq[NOJC
amount of interstitial fibrosis was quantified by histomorphometry. Formalin-fixed, fo
5!d@Nv
paraffin-embedded sections were cut onto coated glass slides. Following heat-induced ?5
+=
antigen retrieval, sections were incubated at 20°C overnight with either anti-CD20 9 s>JdAw?
primary antibody or anti-CD3 primary antibody, both at 1:1000 dilution (Dako, Canada M~wJe@bc
Inc, Mississauga, Ontario, Canada). After rinsing all sections, pretreatment with 3% 8-po|
hydrogen peroxide was performed to prevent endogenous peroxidase activation. Sections hDfsqSK0 /
were incubated with a secondary rabbit anti-mouse antibody linked with avidin–biotin {
\Q'eL8
complex. Sections were counterstained with hematoxylin and examined by light _STN ^
microscopy. y=q\1~] Z
The HACA assay is a proprietary bridging enzyme-linked immunosorbent assay nC2A&n