18 kPX2e h
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number )C01fZhD
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The ]1|Ql*6y,
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at :UDn^(#
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The P2Qyz}!wo
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 'UvS3]bSYW
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, n:^"[Le
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive 4>"cc@8&~
control for Ca2+ induction. The data thus obtained was analyzed with the software Win ic0v*Y$
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on U_}A{bFG
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 ADv"_bB:h
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were 78a-3){
harvested and fractionated as detailed above, and the S-100 fractions were assessed by (&P0la1
immunoblotting for presence or absence of annexin I. bhqq
Mouse bone marrow transduction and transplantation. 2htA7V*dD
Retrovirus-mediated transduction of mouse bone marrow cells was done by published 4/`h@]8P
methods49. Prestimulated low-density bone marrow cells were infected with high-titer {r].SrW9s9
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated lR@i`)'?U
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing +F7<5YW&(
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by +i`Q 7+d
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) 4QjWZ Wl
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone 1t\b a1x
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis C'8!cPFVv
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of uFm+Y]h
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was L*UV
confirmed by immunoblot analysis. 1~R$$P11[9
Determination of renal morphology oxMUW<gYd
Kidney slices were postfixed in buffered 2% OsO4, dehydrated, and embedded in an #j^('K|
Araldite-EM bed 812 mixture. Large sections were cut perpendicular to the renal capsule, `
Y{>2UFX
containing cortex, and medulla. Thin (1 m) sections were analyzed in a blinded manner sw41wj
for morphologic alterations, as previously detailed ?y>Y$-v/C
Patient population %K%^ ]{
Patients included in the study met the following criteria: (1) biopsy-proven IMN; (2) X/E7o92\
creatinine clearance 30 ml per min per 1.73 m2; and (3) persistent proteinuria >5 g per %qS]NC
24 h despite treatment with an HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) _}I(U?Q-C
reductase inhibitor, an ACEi, and/or ARB at maximal tolerated dose for at least 4 months. XdGpW
The Mayo Institutional Review Board and the Research Ethical Board, University Health 2/yXY_L
Network, University of Toronto approved the study protocol. All patients gave written ?~aZ#%*i8
informed consent. Patients who had been on treatment with prednisone, cyclosporine, or +9CEC1-l
19 I #M%%5e
mycophenolic mofetil within the last 4 months or alkylating agents within the last 6 @cuD8<\i
months were not included in the study. Patients with active infection, diabetes, or a O;X(pE/G
secondary cause of MN (for example, hepatitis B, systemic lupus erythematosus (SLE), fJ)N:q`
medications, malignancies) were also excluded. xH_A@hf;
Treatment a>wfhmr
At enrollment, a low-sodium (<4 g day-1) and low-protein (0.8 g per kg per day of 0W T#6D
high-quality protein) diet was recommended and patients were encouraged to maintain 2PVx++*]C
the same diet throughout the duration of the study. All patients received a similar "t[M'[ `C
conservative treatment regimen that included loop diuretics to control edema, an lQV|U;~D
HMG-CoA reductase inhibitor, and an ACEi combined with an ARB if tolerated. 4meidKw]
-Blockers and non-dihydropyridine calcium channel blockers, in that order, were added NXzU0
when required to control systolic blood pressures to <135 mm Hg in >75% of the )Qh>0T+(
readings. Patients who after a minimum of 4 months of conservative therapy and [_y9"MMwn
maximized Ang II blockade had proteinuria >5 g per 24 h received two i.v. infusions of 8G 0
rituximab at a dose of 1000 mg on days 1 and 15. To minimize infusion reactions, jW-;4e*H=V
patients were premedicated with acetaminophen (1000 mg) and diphenhydramine gw_|C|!P
hydrochloride (50 mg) orally. In addition, methylprednisolone (100 mg, i.v.) was given /635B*g
prior to the first rituximab infusion. B-cell depletion was defined as CD19+ count rr |"r
<5 cells per l at any time and B-cell recovery was defined as CD19+ cell count _9D|u<D
>15 cells per l. Patients treated with rituximab, who at month 6 had proteinuria >3 g per Wgxn`6
24 h and in whom CD19+ B-cell counts had increased to >15 cells per l, received a >f&xJq
second course of rituximab treatment following the same protocol described above. 2HcsQ*H]G
Follow-up Q`
H#
fS~
In all patients, clinical and laboratory parameters including complete blood counts, :
@'fpN
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum mJ)tHv"7
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry {IJ-4>
and at months 1, 3, 6, 9, and 12. Creatinine clearance and protein and creatinine excretion }o-|8P:Y
in the urine were assessed by performing two consecutive 24-h urine collections at each "Dyym<J
time point. Data were considered accurate when urinary creatinine excretion was b.QpHrnhtK
consistent with a complete 24 h collection. The mean of the two measurements was \CcmePTN#x
considered for the analysis. The presence of HACAs was evaluated at baseline and at
F[5S(7M
7
months 3, 6, 9, and 12. Osnyd+dJY
Method / Approach / Study/ Technique _3|6ZO
A discussion is presented of a problem-solving system )AOPiC$jL
To improve the efficiency of the method, the following approach may be applied. sp|q((z{
In order to an investigation was made to find the causes of the 4<u;a46Z#M
Although large collections of rules and equations have been complied, none are generally l{8CISO*
accepted .>P~uZiX!
20 *2`:VFEV
This approach will be explained and discussed thoroughly in the body of the report. */nuv
k
This can be accomplished by D -6
This algorithm to compute the total cost can be described step by step as follows: ?FN9rhAC
The above preliminary analysis has provided important information ^]VcxKU J
Various methods have been proposed for selecting an optimum... B~?c3:6
These concepts have been applied to aVP5%
On the basis of the concept mentioned above, GWgd8x*V
This can be achieved by
V7:\q^$
In addition, tissues were stained for infiltrating lymphocytes (CD20 and CD3), and the ZPlPN;J^1
amount of interstitial fibrosis was quantified by histomorphometry. Formalin-fixed, mH)OB?+lq
paraffin-embedded sections were cut onto coated glass slides. Following heat-induced wazP,9W?
antigen retrieval, sections were incubated at 20°C overnight with either anti-CD20 RK-x?ZYH'
primary antibody or anti-CD3 primary antibody, both at 1:1000 dilution (Dako, Canada yxvjg\!&
Inc, Mississauga, Ontario, Canada). After rinsing all sections, pretreatment with 3% y:.?5KsPI
hydrogen peroxide was performed to prevent endogenous peroxidase activation. Sections *Rq`*D>:U}
were incubated with a secondary rabbit anti-mouse antibody linked with avidin–biotin ^sV|ck
complex. Sections were counterstained with hematoxylin and examined by light ;,rnk-
microscopy. 9<l-NU9 _
The HACA assay is a proprietary bridging enzyme-linked immunosorbent assay LJWTSf"f?
performed at Genentech Inc. that measures the antibody response to rituximab in human 9:-T@u
serum samples. D^u\l
In all patients, clinical and laboratory parameters including complete blood counts, \!<"7=(J{4
electrolytes, serum albumin, B-cell flow cytometry for CD19+ B cells, serum FZ p<|t
immunoglobulin (IgG, IgM, IgA) levels, and a lipid panel were evaluated at study entry Ff<)4`J
and at months 1, 3, 6, 9, and 12. ra:GzkIw
This fact suggests that a new concept 5mavcle{4r
This was accomplished by taking ... b@6:1x
The preparatory stage is very time consuming process. .E$q&7@/j
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v
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not taken this approach, with the exception of X2^_~<I{,
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It is a very tedious process to go through <
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The entire interpretation process is conducted in one's head. -!TcQzHUs
These approaches are sometimes very tedious. Ea]T>4
Several techniques can be used S?4KC^Y5
A polynomial parametric model can be written as [the following]/[follows]: FJwt?3\u5
A xx model is constructed/formulated using xx. yp+F<5o
A xx model represents an xx by its xx. Pm^lr! 3p
A process decision model captures the logic essential to A(+:S"|@
From the equation above, xx is equal to the summation of xx times the ... hnzNP\$U
]
21 1lcnRHO
The validity of a xx model can be checked using Euler's formula. lFfXWNb
Given a model, one can mathematically determine whether ... or ... 5B*qbM
Equations for xx need to be derived and implemented in the system. ^|?1_r
A number of heuristic rules have been developed for |<(t}}X
Optimum .. techniques can be made more reliable by ... so that _-%d9@x
An algorithm based on the characteristic ... is used to determine ^ ulps**e
Euler's formula states the following: M#a&\cqC
The completed model should agree with the formula. KlUqoJ;"
For manufacturing purposes, a detailed and precise model of the object is necessary 4j3oT)+8
Engineering design models are very well defined; therefore, *jF#^=
To keep the domain narrow enough to be implementable, yet wide enough to be useful. <HQ&-j x
Point of View 1;Xgc@
from an implementation standpoint, <xlm
K(
From the point of view of this application, _M5Xk? e=
From this point of view, Zadeh suggested an inference rule named xxx (CRI for short). a)S7}0|R
Information is the meaningful interpretation and correlation of some aggregation of data cy1jZ1)
in order to allow one to make decisions. ?/d!R]3
From a practical point of view, the computational aspects of an FLC require a &Rp"rMeW
simplification of the fuzzy control algorithm. $bGD%9
z
The use of a hammer to insert screws, although partly effective, tends to distort, destroy, 2U9&l1P=
and generally defeat the purpose of using a screw [Kusiak AI Implications for CIM Jqru AW<
p.129] -0 0}if7
Statistical analysis ]MAT2$"le
Data were analyzed by one-way analysis of variance comparing the three conditions irMd
jG
(sham operation, ischemic AKI, and bilateral nephrectomy) at each time point. If mP-2s;q
significant F-statistic from analysis of variance existed, this test was followed by Dunnett &1GUi{I
post hoc multiple comparison procedure with sham operation as the control group. For all Qb`C)Nh:
other comparisons, Student's t-test was used. A P-value of <0.05 was considered as 9#rt:&xo0
statistically significant. \!H{Ks{#R.
Values are expressed as means s.e.m. and significance was evaluated by Mann–Whitney DJP6TFT&G
U test using GraphPad Prism, version 4.0 software (GraphPad Software Inc., San Diego, >n!ni(
CA, USA). Np)aS[9W
All values are expressed as means s.d. Statistical significance (defined as P<0.05) was +v15[^F
evaluated using analysis of variance and Bonferroni t-tests, and the two-tailed Pearson's ~L4eZ
test, where appropriate. Fu)Th|5GZ
Data are expressed as mean s.d., median and interquartile range, or frequencies, as k'+}92
o
appropriate. Variables who deviated from the normal distribution (positively skewed) <k]qH-v4
were log-transformed (log10) before the correlation study. 2p:r`THvS5
Data are represented as means s.e.m. Student's t-test and multiple comparisons with t-test oJY[{-qW
post hoc analysis of variance were used as indicated below, for the comparison of 2k1aX~?
22 rt b* n~
morphological, immunohistological and functional parameters. Statistical significance X\sm[_I
was set at P<0.05. ,{{SI
The primary efficacy parameter was defined as change in urinary protein excretion from JBcY!dy-d
baseline (week 0) to 12 months after treatment. The 12-month changes were tested 8$}OS-
against zero using the paired t-test. Secondary end points included 6-month changes in t(?<#KUB-
protein; the number with PR or CR at 6 or 12 months; and changes in glomerular $TG?4
filtration rate (GFR), serum albumin, and lipid profiles. Study sample size was based on vJCf~'
the desire to have 80% power to detect a drop in urinary protein of at least 2.0 g day-1. a8s4T$
Assuming a two-sided hypothesis test performed at a significance level of 0.05 and an s.d. |}S1o0v{(a
of urinary protein change of 2.5 g, it was determined that 15 patients were required.2 nl@E[yA9[
Definition of remission status is according to the criteria established by Cattran et al.30 J22r v(
CR was defined as proteinuria <0.3 g per 24 h, PR as proteinuria 3 g per 24 h, and a OFy,B
-`A{
>50% reduction in peak proteinuria and non-response as <50% reduction in peak >q(6,Mmb
proteinuria. Any patient reaching a CR or PR was considered a treatment success nj2gs,k
The statistical significance of differences for the mean values of cytokine concentration R6@uM<