18 OtVRhR3>
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number vR)f'+_Nz
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The q9h3/uTv
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at n?[JPG2X
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The nc2=S^Fqu
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 `k.Tfdu)K
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, ){I0
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive ;J>upI
control for Ca2+ induction. The data thus obtained was analyzed with the software Win u{'|/g&
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on X7g@.Oy`
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 F{*h~7D-|
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were Xt.ca,`U
harvested and fractionated as detailed above, and the S-100 fractions were assessed by ]<xzCPB
immunoblotting for presence or absence of annexin I. _u{z$;
Mouse bone marrow transduction and transplantation. zzH^xxg
Retrovirus-mediated transduction of mouse bone marrow cells was done by published v:1DNR4
methods49. Prestimulated low-density bone marrow cells were infected with high-titer _t/~C*=:=
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated -\9K'8 C
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing yp!7^
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by Mp7X+o/
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) `9*
|Y 8:
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone Bc*FH>E
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis Tpd|+60g
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of fjd)/Gg
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was kj>!&W57
confirmed by immunoblot analysis. Q3I^(Ll"L
Determination of renal morphology ,x=S)
t
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an Q@
Ze+IhK`
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, ,(A
$WT@e
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner 2oo/KndU
for morphologic alterations, as previously detailed >i_ #q$o
Patient population g.*DlD%%
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) gkmV;0
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per >/4N :=.h
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) ueyQ&+6r
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. r])V6 ^U
The Mayo Institutional Review Board and the Research Ethical Board, University Health b+CJRB1
Network, University of Toronto approved the study protocol. All patients gave written =<%[P9y
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or _CMNmmp`e
19 i IM\_<?
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 zP&D
months were not included in the study. Patients with active infection, diabetes, or a .b?
Aq^i8
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), f_2(`T#
medications, malignancies) were also excluded. bC/Ql
Treatment
p ,.6sk
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of (6Ciqf8
high-quality protein) diet was recommended and patients were encouraged to maintain .JOZ2QWm<
the same diet throughout the duration of the study. All patients received a similar $^_6,uBM[
conservative treatment regimen that included loop diuretics to control edema, an q
BIekQT
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. fx-8mf3
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added x"n++j
when required to control systolic blood pressures to <135 mm Hg in >75% of the @^Rl{p
readings. Patients who after a minimum of 4 months of conservative therapy and |BUgsE
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of O0FUJGuTS
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions, }<YU4EW
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine "BVp37m;?
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given zW\s{
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count Gr 4v&Mz:
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count B
6z 'Q
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per OD9z7*E@
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a XF+4*),
second course of rituximab treatment following the same protocol described above.
d@p#{ -
Follow-up
Q6RTH
In all patients, clinical and laboratory parameters including complete blood counts, Ta[\BWR2
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum C5~n^I|
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry $}.+}'7$
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion h!ogH >S~
in the urine were assessed by performing two consecutive 24-h urine collections at each "%)g^Atp>
time point. Data were considered accurate when urinary creatinine excretion was ]B UirJ,2
consistent with a complete 24 h collection. The mean of the two measurements was ZSs@9ej
considered for the analysis. The presence of HACAs was evaluated at baseline and at $4^SWT.
months 3, 6, 9, and 12. Lg~C:BNF
Method / Approach / Study/ Technique >[Wjzg
A discussion is presented of a problem-solving system %S"85#R5E
To improve the efficiency of the method, the following approach may be applied. 33EF/k3vW
In order to an investigation was made to find the causes of the l=b!O
Although large collections of rules and equations have been complied, none are generally )AxD|A
accepted Scx!h. \5
20 :SS \2
This approach will be explained and discussed thoroughly in the body of the report. MqB@}!
This can be accomplished by Y+UJV6
This algorithm to compute the total cost can be described step by step as follows: bY2R/FNL=
The above preliminary analysis has provided important information FGx)
?
Various methods have been proposed for selecting an optimum... GTM0Qvf?
These concepts have been applied to *d3-[HwZCL
On the basis of the concept mentioned above, ZX` \so,&,
This can be achieved by mmbe.$73
In addition, tissues were stained for infiltrating lymphocytes (CD20 and CD3), and the |y[I!JdR
amount of interstitial fibrosis was quantified by histomorphometry. Formalin-fixed, [9${4=Kq
paraffin-embedded sections were cut onto coated glass slides. Following heat-induced R\5,H!V9n
antigen retrieval, sections were incubated at 20°C overnight with either anti-CD20 J<;io!
primary antibody or anti-CD3 primary antibody, both at 1:1000 dilution (Dako, Canada U?sHh2*
Inc, Mississauga, Ontario, Canada). After rinsing all sections, pretreatment with 3%
LNWS
hydrogen peroxide was performed to prevent endogenous peroxidase activation. Sections -0d9,,c
were incubated with a secondary rabbit anti-mouse antibody linked with avidin–biotin W|m(Jh[w]
complex. Sections were counterstained with hematoxylin and examined by light ud r\\5
microscopy. 7DJEx~"!2-
The HACA assay is a proprietary bridging enzyme-linked immunosorbent assay \jwG*a
performed at Genentech Inc. that measures the antibody response to rituximab in human 5[jcw`
serum samples. N:)x67,
In all patients, clinical and laboratory parameters including complete blood counts, Uv(R^50>
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum x 4+WZYv3
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry b^$`2m-?@f
and at months 1, 3, 6, 9, and 12. ~i"=:D
This fact suggests that a new concept &SE}5ddC7
This was accomplished by taking ... (<eLj Q
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Test are performed for validity, completeness, and compatibility v`Jt+?I
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There has been an increasing awareness of the potential of using most ..so far made have !0,q[|m
not taken this approach, with the exception of <{kPa_`'
Only a few studies can be found. e"t0 rScA
It is a very tedious process to go through LS[o7 !T(
It is only when .. has been completed that .. may be effected rkjnw@x\
The entire interpretation process is conducted in one's head. ']1n?K=A
These approaches are sometimes very tedious. eL}w{Hlk
T
Several techniques can be used @:}c
(j
A polynomial parametric model can be written as [the following]/[follows]: .G[/4h :.
A xx model is constructed/formulated using xx. lnyb4d/
A xx model represents an xx by its xx. Z\nDR|3
A process decision model captures the logic essential to ^X96yj'?
From the equation above, xx is equal to the summation of xx times the ...
Z_q+Ac{p
21 0SI@`C*1o
The validity of a xx model can be checked using Euler's formula. PR@6=[|d
Given a model, one can mathematically determine whether ... or ... ai_ve[A
Equations for xx need to be derived and implemented in the system. sUG!dwqqd
A number of heuristic rules have been developed for Hh%!4_AMw
Optimum .. techniques can be made more reliable by ... so that J~2SGXH)^?
An algorithm based on the characteristic ... is used to determine f7x2"&?vg
Euler's formula states the following: OXEEpoU?V
The completed model should agree with the formula. Mh)?A/e
For manufacturing purposes, a detailed and precise model of the object is necessary fQfn7FaW_\
Engineering design models are very well defined; therefore, } k2Q
To keep the domain narrow enough to be implementable, yet wide enough to be useful. Pu3oQDldV
Point of View )pHtsd. eP
from an implementation standpoint, 4qid+ [B
From the point of view of this application, sSW'SE?,<
From this point of view, Zadeh suggested an inference rule named xxx (CRI for short). c2P}P* _
Information is the meaningful interpretation and correlation of some aggregation of data (GC]=
in order to allow one to make decisions. X!]v4ma`
From a practical point of view, the computational aspects of an FLC require a -Uo11'{
simplification of the fuzzy control algorithm. lbHgxZ
The use of a hammer to insert screws, although partly effective, tends to distort, destroy, ~+Gh{,f
and generally defeat the purpose of using a screw [Kusiak AI Implications for CIM *~^6
3Nx!
p.129] 83vMj$P
Statistical analysis 3}|[<^$
Data were analyzed by one-way analysis of variance comparing the three conditions xgk~%X%K
(sham operation, ischemic AKI, and bilateral nephrectomy) at each time point. If ? "I %K%
significant F-statistic from analysis of variance existed, this test was followed by Dunnett 2^o7 ^S
post hoc multiple comparison procedure with sham operation as the control group. For all m]n2wmE3n
other comparisons, Student's t-test was used. A P-value of <0.05 was considered as 9B0ON*`
statistically significant.
J0!V (
Values are expressed as means s.e.m. and significance was evaluated by Mann–Whitney -EiTP:A
U test using GraphPad Prism, version 4.0 software (GraphPad Software Inc., San Diego, _WEJ,0*#'
CA, USA). gcA:Q4
All values are expressed as means s.d. Statistical significance (defined as P<0.05) was u9"=t
evaluated using analysis of variance and Bonferroni t-tests, and the two-tailed Pearson's O*~,L6# }
test, where appropriate. /QQRy_Z1)
Data are expressed as mean s.d., median and interquartile range, or frequencies, as NH/A`Wm
appropriate. Variables who deviated from the normal distribution (positively skewed) ;I4vPh5Q
were log-transformed (log10) before the correlation study. B7f<XBU6>
Data are represented as means s.e.m. Student's t-test and multiple comparisons with t-test z8X7Y
>+SA
post hoc analysis of variance were used as indicated below, for the comparison of R/~!km
22 Rt#QW*h\|i
morphological, immunohistological and functional parameters. Statistical significance _KM?
?&
was set at P<0.05. :Qge1/
The primary efficacy parameter was defined as change in urinary protein excretion from uY5|Nmiu
baseline (week 0) to 12 months after treatment. The 12-month changes were tested _WZx].|A=
against zero using the paired t-test. Secondary end points included 6-month changes in LA Crg
protein; the number with PR or CR at 6 or 12 months; and changes in glomerular FsED9+/m
filtration rate (GFR), serum albumin, and lipid profiles. Study sample size was based on =
2 HY]H
the desire to have 80% power to detect a drop in urinary protein of at least 2.0 g day-1. TQ(q[:>
Assuming a two-sided hypothesis test performed at a significance level of 0.05 and an s.d. z/J?!ee
of urinary protein change of 2.5 g, it was determined that 15 patients were required.2 :TTq
Definition of remission status is according to the criteria established by Cattran et al.30 2PQBUq
CR was defined as proteinuria <0.3 g per 24 h, PR as proteinuria 3 g per 24 h, and a M`&t=0D
>50% reduction in peak proteinuria and non-response as <50% reduction in peak 4~}NB%,
proteinuria. Any patient reaching a CR or PR was considered a treatment success 3
`_/h' ~
The statistical significance of differences for the mean values of cytokine concentration ^Gd1T
and T cell proliferation was determined with Student's t-test. Differences with a P value g4^-B
of less than 0.05 were considered significant. @(#vg\UH
Statistical significance was determined by Student's t-test. Data with a P value of less gkmof^
than 0.05 were considered significant. 5ngs1ZF@
In vitro and in vivo experiments were analyzed by the two-tailed Student's t-test, with P #mU\8M,
values less than 0.05 considered statistically significant. @|:fm()
<
The significance of differences among groups was determined by Student's t-test and Qf_N,Bq{a
one-way analysis of variance (SigmaStat software; Jandel). r-T1^u
Comparisons T
t~4'{Bc
As well, the pros and cons of these representations from a process planning point of view /H<{p$Wd
will be discussed. 1`?o#w
The method of using xx to implement xx described by Zadeh (1973) appeared more NV./p`k
suitable YW7w>}aW
As discussed [in the previous section]/[preciously], ;}Jv4Z
Relation I;@q`Tm
We can not invert F' directly because it defines a many-to-one mapping. 5 SQ!^1R 9
The relationships appear very complicate Py*WHHO
Lifting tasks involve complex and imprecise relationship between the task variables and +^9^)Ur|
the human operator's characteristics. {e&fB