18 l (%1jC8
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number +@:x!q|^
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The +H
Usz?
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at ,u
g@f-T
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The V/;B3t~f
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 {>%&(
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, e*n@j
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive -S+zmo8
control for Ca2+ induction. The data thus obtained was analyzed with the software Win M:6"H%h,W
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on 8Bg;Kh6B
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 )];K .zP
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were [66!bM&
harvested and fractionated as detailed above, and the S-100 fractions were assessed by 0{[,E.
immunoblotting for presence or absence of annexin I. Q1l '7N
Mouse bone marrow transduction and transplantation. .y,0[i V
N
Retrovirus-mediated transduction of mouse bone marrow cells was done by published ff1c/c/
methods49. Prestimulated low-density bone marrow cells were infected with high-titer T${Q.zHY[!
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated <1COZ)
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing 9?3&?i2-
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by :<#nTh_@\'
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) gOOPe5+ J
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone _H=Uwi_g
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis |
qZ1|
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of [R7Y}k:9U
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was '-/xyAzS
confirmed by immunoblot analysis. R+,u^;\
Determination of renal morphology R n*
L
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an x
7x\Y(@
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, nL.<[]r
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner 9v!1V,`j"
for morphologic alterations, as previously detailed N<KS(@v
y
Patient population M|(Q0 _8
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) _M5|Y@XN-
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per H_<C!OgR
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) L4|`;WP
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. '1)$'
The Mayo Institutional Review Board and the Research Ethical Board, University Health B|AV$N*
Network, University of Toronto approved the study protocol. All patients gave written fCobzDy
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or fku<,SV$O4
19 >q1L2',pK
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 GU8sO@S5
#
months were not included in the study. Patients with active infection, diabetes, or a @Sbe^x
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), @)&=%
medications, malignancies) were also excluded. T+k{W6
Treatment dIBE!4 V[
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of N;j)k;
high-quality protein) diet was recommended and patients were encouraged to maintain E6gI,f/p0X
the same diet throughout the duration of the study. All patients received a similar BV upDGh3
conservative treatment regimen that included loop diuretics to control edema, an V2|aN<Sx<
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. {"QNJq#:
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added j578)!aJ
when required to control systolic blood pressures to <135 mm Hg in >75% of the O[)kboY
readings. Patients who after a minimum of 4 months of conservative therapy and s;vHPUB\n
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of N
yj( 0W
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions, !XCm>]R
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine ;Q*or2"
!
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given thM4vq
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count \\dMy9M-
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count g($DdKc|g
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per >P@H#=
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a a^zibPG
second course of rituximab treatment following the same protocol described above. (/j/>9iro
Follow-up 1\>^m
In all patients, clinical and laboratory parameters including complete blood counts, 4f'V8|QM{
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum }h!f
eP
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry [^e%@TV>d
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion ~W+kiTsD?
in the urine were assessed by performing two consecutive 24-h urine collections at each g8xQ|px
time point. Data were considered accurate when urinary creatinine excretion was VAf1 " )pC
consistent with a complete 24 h collection. The mean of the two measurements was ^xh ;
considered for the analysis. The presence of HACAs was evaluated at baseline and at )ZqTwEr@[
months 3, 6, 9, and 12. `_RTw5{
Method / Approach / Study/ Technique Xudg2t)+K
A discussion is presented of a problem-solving system 1Z~)RJ<D
To improve the efficiency of the method, the following approach may be applied. .=;3d~.]
In order to an investigation was made to find the causes of the -~30)J=e`
Although large collections of rules and equations have been complied, none are generally M
.JoHH
accepted E}p&2P+MR
20 U4-g^S[
This approach will be explained and discussed thoroughly in the body of the report. f[a}aZ9)
This can be accomplished by vUU9$x
This algorithm to compute the total cost can be described step by step as follows: <55g3>X
The above preliminary analysis has provided important information Vz~nT
Various methods have been proposed for selecting an optimum... -[!P!
d=
These concepts have been applied to R0vI bFwj
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This can be achieved by %$Z7x\_
In addition, tissues were stained for infiltrating lymphocytes (CD20 and CD3), and the Hp|_6hO 2
amount of interstitial fibrosis was quantified by histomorphometry. Formalin-fixed, `_{`l4i5
paraffin-embedded sections were cut onto coated glass slides. Following heat-induced /[)qEl2]K
antigen retrieval, sections were incubated at 20°C overnight with either anti-CD20 H_ox_
u}
primary antibody or anti-CD3 primary antibody, both at 1:1000 dilution (Dako, Canada pxf$1
Inc, Mississauga, Ontario, Canada). After rinsing all sections, pretreatment with 3% H77"
hydrogen peroxide was performed to prevent endogenous peroxidase activation. Sections {v2|g
were incubated with a secondary rabbit anti-mouse antibody linked with avidin–biotin ^8Q62
complex. Sections were counterstained with hematoxylin and examined by light ?)X,0P'
microscopy. U1RpLkibQ
The HACA assay is a proprietary bridging enzyme-linked immunosorbent assay >W`4aA
performed at Genentech Inc. that measures the antibody response to rituximab in human `~;rblo;
serum samples. C@W"yYt
In all patients, clinical and laboratory parameters including complete blood counts, R Yl>
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum nwaxz
>;
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry <5jzl
and at months 1, 3, 6, 9, and 12. K7Wk6Aw
This fact suggests that a new concept wG<(F}VX
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not taken this approach, with the exception of mAW,?h
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The entire interpretation process is conducted in one's head. L^4-5`gj
These approaches are sometimes very tedious. ;cz|ss=
Several techniques can be used Ch%m
A polynomial parametric model can be written as [the following]/[follows]: Bk\Gj`"7
A xx model is constructed/formulated using xx. 6]pX>Xho
A xx model represents an xx by its xx. VZ](uF BY
A process decision model captures the logic essential to F]t(%{#W
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21 @L:>!
<
The validity of a xx model can be checked using Euler's formula. {$^DMANDx
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Equations for xx need to be derived and implemented in the system. j$Je6zq0x
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Optimum .. techniques can be made more reliable by ... so that ?8mlZ
X9C
An algorithm based on the characteristic ... is used to determine (q7
Ry4-
Euler's formula states the following: &0 BdUU+:<
The completed model should agree with the formula. ?k|H3;\
For manufacturing purposes, a detailed and precise model of the object is necessary Q\,o:ZU_
Engineering design models are very well defined; therefore, p/
>`[I
To keep the domain narrow enough to be implementable, yet wide enough to be useful. d{de6 `
Point of View w7n373y%
from an implementation standpoint, .pvV1JA'
From the point of view of this application,
5rV((
From this point of view, Zadeh suggested an inference rule named xxx (CRI for short). H7kPM[
Information is the meaningful interpretation and correlation of some aggregation of data cFF*Z=L_
in order to allow one to make decisions. ^@_m "^C
From a practical point of view, the computational aspects of an FLC require a {gaai
simplification of the fuzzy control algorithm. rUjdq/I:Z
The use of a hammer to insert screws, although partly effective, tends to distort, destroy, Vax^8 -
and generally defeat the purpose of using a screw [Kusiak AI Implications for CIM eVcANP
p.129] 7 #=}:3c
Statistical analysis ')$NfarQ.
Data were analyzed by one-way analysis of variance comparing the three conditions PSmfiaThwo
(sham operation, ischemic AKI, and bilateral nephrectomy) at each time point. If 45H!;Qsk
significant F-statistic from analysis of variance existed, this test was followed by Dunnett
G^1b>K
post hoc multiple comparison procedure with sham operation as the control group. For all TV}}dw
other comparisons, Student's t-test was used. A P-value of <0.05 was considered as m%8qZzqk
statistically significant. VNtPKtx\
Values are expressed as means s.e.m. and significance was evaluated by Mann–Whitney <d7V<&@o=
U test using GraphPad Prism, version 4.0 software (GraphPad Software Inc., San Diego, !"TZ:"VZU
CA, USA). fXQiNm[P
All values are expressed as means s.d. Statistical significance (defined as P<0.05) was 1SV^ ){5I
evaluated using analysis of variance and Bonferroni t-tests, and the two-tailed Pearson's N|2y"5
test, where appropriate. )2E%b+"
Data are expressed as mean s.d., median and interquartile range, or frequencies, as ;bX4(CMe
&
appropriate. Variables who deviated from the normal distribution (positively skewed) \ U-vI:J_
were log-transformed (log10) before the correlation study. @oG)LT
Data are represented as means s.e.m. Student's t-test and multiple comparisons with t-test 2~;&g?T6
post hoc analysis of variance were used as indicated below, for the comparison of bxXiQa
22 =qvZpB7ZZ
morphological, immunohistological and functional parameters. Statistical significance i(6J>^I
was set at P<0.05. "MiD8wX-
The primary efficacy parameter was defined as change in urinary protein excretion from wp.TfKxw
baseline (week 0) to 12 months after treatment. The 12-month changes were tested ~
-F?Mc
against zero using the paired t-test. Secondary end points included 6-month changes in )-/gLZsx
protein; the number with PR or CR at 6 or 12 months; and changes in glomerular ^`qPs/b
filtration rate (GFR), serum albumin, and lipid profiles. Study sample size was based on !@
YXZ
the desire to have 80% power to detect a drop in urinary protein of at least 2.0 g day-1. K.SeK3(
Assuming a two-sided hypothesis test performed at a significance level of 0.05 and an s.d. }+Vv0jX|V
of urinary protein change of 2.5 g, it was determined that 15 patients were required.2 w9#R'
Definition of remission status is according to the criteria established by Cattran et al.30 BZF,=v
CR was defined as proteinuria <0.3 g per 24 h, PR as proteinuria 3 g per 24 h, and a |[cdri^?D
>50% reduction in peak proteinuria and non-response as <50% reduction in peak 3#<*k>1G?
proteinuria. Any patient reaching a CR or PR was considered a treatment success ?Cci:Lin
The statistical significance of differences for the mean values of cytokine concentration p`+VrcCBOd
and T cell proliferation was determined with Student's t-test. Differences with a P value y>(rZ^y&
of less than 0.05 were considered significant. 4^!4eyQ^
Statistical significance was determined by Student's t-test. Data with a P value of less MPRO
!45Z
than 0.05 were considered significant. ]X" / yAn
In vitro and in vivo experiments were analyzed by the two-tailed Student's t-test, with P Wtv#h~jy9
values less than 0.05 considered statistically significant. >v0 :qN7|
The significance of differences among groups was determined by Student's t-test and nJVp.*S
one-way analysis of variance (SigmaStat software; Jandel). PX 3
Comparisons u+% tPe
As well, the pros and cons of these representations from a process planning point of view M<