18 5jy>)WqK
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number sj. eJX"z
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The ffm19 B=
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at G~4 ^`[elB
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The &.7\{q\(
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 xvp{F9~qT
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, {(7.X4\x
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive r-Z'
control for Ca2+ induction. The data thus obtained was analyzed with the software Win F6Q #{Ufq
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on P).
@o.xl
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 aQFYSl
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were _E'M(.B<
harvested and fractionated as detailed above, and the S-100 fractions were assessed by pBL{DgX
immunoblotting for presence or absence of annexin I. s o~p+]
Mouse bone marrow transduction and transplantation. -+Q,xxu
Retrovirus-mediated transduction of mouse bone marrow cells was done by published [mWo&Ph[-
methods49. Prestimulated low-density bone marrow cells were infected with high-titer 1P2%n[y
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated 8%
1hfj
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing 8%`Sx[
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by {_[l,tdZ
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) 6Mk@,\1
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone SC|cCK hqi
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis u%}vTCg*p
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of
*~ &W?i
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was ZQ20IY|,
confirmed by immunoblot analysis. C
7+TnJ
Determination of renal morphology S~6<'N&[
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an 4,<~t>M1
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, fG,qax`:c
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner OvK_CN{
for morphologic alterations, as previously detailed }#E4t3
Patient population 2d3wQ)2
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) `b7
o
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per orHVL 2
KK
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) )Fm
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. Yhlk#>I
The Mayo Institutional Review Board and the Research Ethical Board, University Health Xq)'p8C?
Network, University of Toronto approved the study protocol. All patients gave written s#qq%
@
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or 9d2$F9]:o
19 r`7`f xe
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 }%XNB1/`
months were not included in the study. Patients with active infection, diabetes, or a #x%O0
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), HQUL?URt
medications, malignancies) were also excluded. 2G'G45Q
Treatment nK96A.B%p
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of xMuy[)b
high-quality protein) diet was recommended and patients were encouraged to maintain }'X}!_9w>
the same diet throughout the duration of the study. All patients received a similar &=7ur
conservative treatment regimen that included loop diuretics to control edema, an h2BD?y
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. _D+7w'8h
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added
94lm
sE
when required to control systolic blood pressures to <135 mm Hg in >75% of the !.w S+
readings. Patients who after a minimum of 4 months of conservative therapy and 6"=e+V@
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of }h]:I'R!
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions, 9g*~X;`2
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine Tw`l4
S&
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given )8N/t6Q
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count .#}SK!"B
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count iL\\JuY
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per {Rz`)qqE
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a zf S
<X
second course of rituximab treatment following the same protocol described above. #Gg^fm
Follow-up [ z&y]~
In all patients, clinical and laboratory parameters including complete blood counts, 8t7hN?,t
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum yT,UM^'
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry $zbg
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion ; 6zu!
in the urine were assessed by performing two consecutive 24-h urine collections at each xFvSQ`sp
time point. Data were considered accurate when urinary creatinine excretion was onmO>q*
consistent with a complete 24 h collection. The mean of the two measurements was EgO4:8$h
considered for the analysis. The presence of HACAs was evaluated at baseline and at {C6
Yr9
months 3, 6, 9, and 12. Fd91Y
Method / Approach / Study/ Technique =o-qu^T^u
A discussion is presented of a problem-solving system LTCjw_<7
To improve the efficiency of the method, the following approach may be applied. |On6?5((e
In order to an investigation was made to find the causes of the
KA<
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accepted Zsuh 8t
20 ILpB:g
This approach will be explained and discussed thoroughly in the body of the report. (sl~n_<ds8
This can be accomplished by l&yR-FJ7KY
This algorithm to compute the total cost can be described step by step as follows: JxWH
rsh[
The above preliminary analysis has provided important information 4EELaP|%
Various methods have been proposed for selecting an optimum... i a|F
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This can be achieved by }I;W
In addition, tissues were stained for infiltrating lymphocytes (CD20 and CD3), and the +X&B'
amount of interstitial fibrosis was quantified by histomorphometry. Formalin-fixed, -g>27EI5
paraffin-embedded sections were cut onto coated glass slides. Following heat-induced H;CGLis
antigen retrieval, sections were incubated at 20°C overnight with either anti-CD20 B%t^QbU #\
primary antibody or anti-CD3 primary antibody, both at 1:1000 dilution (Dako, Canada jv7zvp
Inc, Mississauga, Ontario, Canada). After rinsing all sections, pretreatment with 3% Jk~T.p?tF
hydrogen peroxide was performed to prevent endogenous peroxidase activation. Sections e9:l
were incubated with a secondary rabbit anti-mouse antibody linked with avidin–biotin l^vq'<kI
complex. Sections were counterstained with hematoxylin and examined by light g7H;d
microscopy. O
o0$n]*;W
The HACA assay is a proprietary bridging enzyme-linked immunosorbent assay x?%vqg^r
performed at Genentech Inc. that measures the antibody response to rituximab in human rd"]$_P8O
serum samples. GM%|mFqeu
In all patients, clinical and laboratory parameters including complete blood counts, F3qi$ 3HM
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum ecFI"g
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry 0 #q_LB
and at months 1, 3, 6, 9, and 12. 0>'1|8+`(z
This fact suggests that a new concept *=I#VN*_<.
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not taken this approach, with the exception of *s!8BwiE
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The entire interpretation process is conducted in one's head. XJQ[aU"[]N
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A polynomial parametric model can be written as [the following]/[follows]: 9 ROKueP
A xx model is constructed/formulated using xx. |S4yol
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O:j
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21 gy>2=d
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Euler's formula states the following: B4{A(-Tc
The completed model should agree with the formula. U%KoG-#
For manufacturing purposes, a detailed and precise model of the object is necessary P= ]ZXj[
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To keep the domain narrow enough to be implementable, yet wide enough to be useful.
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Point of View 8Mp
from an implementation standpoint, ."h;H^5
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P=
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V$H
in order to allow one to make decisions. #}A!Bk
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simplification of the fuzzy control algorithm. #7g~Um%p
The use of a hammer to insert screws, although partly effective, tends to distort, destroy, "#p)Z{v"!
and generally defeat the purpose of using a screw [Kusiak AI Implications for CIM 5v?6J#]2
p.129] "r|O /
Statistical analysis y[}BFUy
Data were analyzed by one-way analysis of variance comparing the three conditions lk+)-J-lj'
(sham operation, ischemic AKI, and bilateral nephrectomy) at each time point. If gsAcn
significant F-statistic from analysis of variance existed, this test was followed by Dunnett 6
bU/IVP
post hoc multiple comparison procedure with sham operation as the control group. For all :1AOund
other comparisons, Student's t-test was used. A P-value of <0.05 was considered as Bg34YmZ
statistically significant. <x1(}x:u`
Values are expressed as means s.e.m. and significance was evaluated by Mann–Whitney *ZY{^f
U test using GraphPad Prism, version 4.0 software (GraphPad Software Inc., San Diego, 8IVKS>
CA, USA). /.}&yRR
All values are expressed as means s.d. Statistical significance (defined as P<0.05) was VGe/;&1h
evaluated using analysis of variance and Bonferroni t-tests, and the two-tailed Pearson's [y'jz~9c
test, where appropriate. S__ o#nf`%
Data are expressed as mean s.d., median and interquartile range, or frequencies, as 8XT
Vpf4
appropriate. Variables who deviated from the normal distribution (positively skewed) _tYt<oB~%
were log-transformed (log10) before the correlation study. *%g*Np_P
Data are represented as means s.e.m. Student's t-test and multiple comparisons with t-test ;B
tRDKn
post hoc analysis of variance were used as indicated below, for the comparison of fOrqY,P'
22 lMlXK4-
morphological, immunohistological and functional parameters. Statistical significance =WN6Fj`
was set at P<0.05. kkqrlJO|
The primary efficacy parameter was defined as change in urinary protein excretion from F-2HE><
+
baseline (week 0) to 12 months after treatment. The 12-month changes were tested & {B,m%G
against zero using the paired t-test. Secondary end points included 6-month changes in A87Tyk2Pi
protein; the number with PR or CR at 6 or 12 months; and changes in glomerular jp2l}C
filtration rate (GFR), serum albumin, and lipid profiles. Study sample size was based on o.sa?*
the desire to have 80% power to detect a drop in urinary protein of at least 2.0 g day-1. #E*jX-JT
Assuming a two-sided hypothesis test performed at a significance level of 0.05 and an s.d. Kf(% aDYq
of urinary protein change of 2.5 g, it was determined that 15 patients were required.2 '"=C^f
Definition of remission status is according to the criteria established by Cattran et al.30 3WaYeol`
CR was defined as proteinuria <0.3 g per 24 h, PR as proteinuria 3 g per 24 h, and a 2,dGRf
>50% reduction in peak proteinuria and non-response as <50% reduction in peak #B>Hq~ vrC
proteinuria. Any patient reaching a CR or PR was considered a treatment success ptrLnJ|%
The statistical significance of differences for the mean values of cytokine concentration @{~x:P5g
and T cell proliferation was determined with Student's t-test. Differences with a P value tpA7"JD
of less than 0.05 were considered significant. KWU#Swa`
Statistical significance was determined by Student's t-test. Data with a P value of less V .$<