178

Induction of Apoptosis in Normal Human Renal Tubular Epithelial Cells by

Escherichia coli Shiga Toxins 1 and 2
Nobutaka Kiyokawa, Tomoko Taguchi, Tetsuya Mori, Department of Pathology, and Department of Infectious Diseases
Hiroshi Uchida, Norihide Sato, Tae Takeda, Research, National Children’s Medical Research Center, Tokyo, Japan
and Junichiro Fujimoto

The cytotoxicity of Shiga toxin (Stx) 1 and Stx2 produced by Escherichia coli to human renal
cortical epithelial cells (HRCEC) in primary culture was investigated. HRCEC express CD24, the
marker of renal distal tubules, as well as globotriaosyl ceramide/CD77, the receptor for Stxs. Binding
of Stxs to HRCEC was confirmed by positive staining with specific antibodies to Stxs. Treatment
of HRCEC with Stxs induced rapid cell death, which was reversed in the presence of neutralizing
antibody specific for Stx. DNA fragmentation was found to be accompanied by Stx-mediated cell
death in HRCEC, indicating that apoptosis was part of the process. These data and previous reports

Downloaded from http://jid.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015

indicate that a variety of renal cell types, including tubular epithelial cells as well as glomerular
capillary endothelial cells, may be targets for Stx-mediated apoptosis, which could contribute to the
pathogenesis of hemolytic-uremic syndrome caused by Stx-producing E. coli infection.

Recent worrisome increases in the incidences of infections
due to Shiga toxin (Stx) – producing strains of Escherichia coli
(STEC) throughout the world have attracted the attention of
numerous public health workers. The major outbreaks in the
western part of the United States, Germany, and Japan are the
latest cases in point. The major symptom of STEC infection
is bloody diarrhea. However, a more serious problem associated
with this infection is the frequent development of a fatal sys-
temic complication called hemolytic-uremic syndrome (HUS)
[1].
HUS, characterized by the triad of hemolytic anemia, acute
renal failure, and thrombocytopenia, remains the leading cause
of acute renal failure in childhood [2]. Although Stxs have
been postulated since the early 1980s to be the substance re-
sponsible for HUS development [1], in vivo kinetics of Stxs
as well as the pathogenesis of HUS are still unclear. However,
on the basis of histopathologic findings [3] and in vitro experi-
ments [4 – 7], endothelial damage in the glomeruli and arterioles
of the kidney induced by Stx is believed to play a crucial
role in the pathogenesis of Stx-mediated HUS [4, 8]. A few
observations on the correlation between apoptosis of glomeru-
lar endothelial cells and sporadic (non – Stx-mediated) HUS or
Received 1 December 1997; revised 10 February 1998.
Financial support: Ministry of Health and Welfare (Health Sciences Research
Grants, Grant for Pediatric Research [9C-04, 9C-05], Grant-in Aid for Cancer
Research [5-24, 9-10]; Entrustment of Research Programme of the Foundation
for Promotion of Cancer Research in Japan; Program for Promotion of Funda-
mental Studies in Health Sciences of the Organization for Drug ADR Relief,
R & D Promotion and Product Review of Japan; Foundation for Promotion of
Cancer Research (Research Resident Fellowship to N.S.).
Reprints or correspondence: Dr. Nobutaka Kiyokawa, Department of Pathol-
ogy, National Children’s Medical Research Center, 3-35-31, Taishido, Seta-
gaya-ku, Tokyo 154-8509, Japan (nkiyokawa@nch.go.jp).
The Journal of Infectious Diseases 1998;178:178–84
q 1998 by the Infectious Diseases Society of America. All rights reserved.
0022–1899/98/7801–0022$02.00
a related disorder, thrombotic thrombocytopenic purpura, may
support this idea [9 – 11].
On the other hand, several studies demonstrated that renal
tubular epithelial cells are another possible primary target for
Stxs [12 – 18]. First, in a murine model, injection of Stxs and
oral administration of STEC have been reported to damage the
cortical tubular epithelium while glomeruli remain unaffected
[12 – 14]. Second, the expression of the functional receptor for
Stxs, globotriaosyl ceramide (Gb3)/CD77 [19 – 21], and the
binding of Stxs has been demonstrated on renal tubular epithe-
lial cells [15, 16]. Third, we have observed marked elevation
of urinary N-acetyl glucosaminidase and b2-microglobulin,
both of which are specific markers of tubular function, in the
acute stage of HUS associated with STEC infection [17].
In connection with the above reports, we previously demon-
strated that Stxs are cytotoxic to the renal carcinoma line
ACHN, an in vitro model of human renal tubular epithelial
cells, which is 106- to 107-fold more sensitive to Stxs than are
HUVEC [17]. More interestingly, we have recently shown that
apoptosis is involved in Stx-mediated cytotoxicity against
ACHN cells [18].
To address whether these observations in ACHN represent
the nature of normal tubular epithelial cells in the kidney, we
examined the effect of Stxs on normal human renal cortical
epithelial cells (HRCEC) by using a primary culture system.
We used HRCEC as an in vitro model of distal tubular epithelial
cells in the kidney and investigated the mechanism of the cyto-
toxicity of Stx1 and Stx2.
Materials and Methods
Shiga toxins, cells, antibodies, and cytokines. Stx1 and Stx2
were prepared as described previously [22, 23]. Normal HRCEC
in primary culture, derived from a 24-year-old volunteer, were
purchased from Clonetics (Walkersville, MD) and maintained at
377C in a humidified 5% CO2 atmosphere. The monoclonal anti-

/ 9d4a$$jy16 05-27-98 00:20:51 jinfa UC: J Infect

JID 1998;178 (July) Shiga Toxins Induce Apoptosis in Renal Tubules 179

bodies used in this study were CD10 (IF-6 [24]), CD24 (L30 [25]),
CD34 (QBEnd10; Coulter/Immunotec, Westbrook, MA), Apo2.7
(Coulter), CD77 (38.13, Coulter; and 1A4, gift of Reiji Kannagi,
Laboratory of Experimental Pathology, Research Institute, Aichi
Cancer Center, Nagoya, Japan), anti-Stx1 (13C4, ATCC CRL 1794
[26]), and anti-Stx2 (11E10, ATCC CRL 1907; and 11F11, ATCC
CRL 1908 [27]). Fluorescein-conjugated and enzyme-conjugated
secondary antibodies were purchased from Jackson Laboratory
(West Grove, PA).
Immunofluorescence study. Cell suspensions of HRCEC were
produced by treating the cells with a non-enzymatic cell dissocia-
tion solution (Sigma-Aldrich, St. Louis) for 5 min, followed by
pipetting. The cells were then stained with monoclonal antibodies
and analyzed by flow cytometry (EPICS Profile and EPICS-XL;
Coulter) as described previously [24]. To detect Stx binding, 106

Downloaded from http://jid.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015

cells in suspension were incubated with and without 100 ng/mL
Stx1 or Stx2 for 1 h at 47C followed by intensive washing before
staining. To detect 7A6 antigen, which is located in the cytoplasm,
Stx-treated cells were harvested and fixed, followed by permeabili-
zation before staining with phycoerythrin-Cy5–conjugated
APO2.7 antibody, as described previously [28].
Immunohistochemical analysis. For immunohistochemical
analysis, cells grown on tissue culture chamber slides (Lab-Tek;
Miles Laboratories, Naperville, IL) were washed with PBS and air
dried, followed by fixation with 100% acetone for 15 min at 47C.
Immunoperoxidase staining was done as described previously [29].
To detect binding of Stxs, cells fixed on glass slides were incubated
with 100 ng/mL Stx1 or Stx2 for 1 h at room temperature before
staining with specific antibody.
MTT assay. To assess growth and viability, cells were plated
on 96-well plates (Corning, Corning, NY) at a concentration of
104 cells in 100 mL of complete medium/well, with and without
Stxs. After incubation for the indicated periods, MTT assays were
done as described previously [30]. Each experiment was done in
triplicate, and averages were calculated.
DNA fragmentation assay. Cells were assayed for DNA ladder
formation by gel electrophoresis. After treatment with and without
Stxs, DNAs were extracted from the cells, separated in 1% agarose
gels by electrophoresis, and examined under UV light as described
previously [31].
DNA fragmentation was also investigated in situ by the TUNEL
method using a in situ apoptosis detection kit (TACS 2 TdT[TBL]; Figure 1. Analysis of antigen expression on surfaces of HRCEC.
Trevigen, Gaithersburg, MD). Experiments were done according A, HRCEC in suspension were stained with specific monoclonal anti-
to the manufacturer’s protocol. bodies and analyzed by flow cytometry. Histogram obtained was
To evaluate relative DNA contents, cells were stained with pro- superimposed on that of negative control (CNT; cells stained with
pidium iodide (Sigma), then analyzed by flow cytometry as de- isotype-matched control immunoglobulin). x axis, fluorescence inten-
scribed previously [32]. sity; y axis, relative cell number. B, HRCEC were grown on chamber
slides. After fixation with 100% cold acetone, cells were immunohis-
tochemically stained with specific monoclonal antibodies.

Results

Characterization of cell surface antigens expressed on
HRCEC in primary culture. It has been reported that the
epithelial components of renal tubules can be classified ac-
cording to the expression pattern of hematopoietic cell surface
antigens [29, 33, 34]. Thus, we first characterized cell surface
antigens expressed on HRCEC by using antibodies against CD
leukocyte antigens. As shown in figure 1A, HRCEC were
strongly positive for CD24 on flow cytometry but negative for
CD10 and CD34. HRCEC were also found to express low
levels of Gb3/CD77, the functional receptor for Stxs. The pat-
tern of the histograms, which depict rather wide peaks, is indic-
ative of a less-than-homogeneous cell population.
The expressions of CD24 and CD77 were also confirmed
by immunohistochemistry (figure 1B). On the basis of the dis-

/ 9d4a$$jy16 05-27-98 00:20:51 jinfa UC: J Infect

180 Kiyokawa et al. JID 1998;178 (July)

Cytotoxic effect of Stxs on HRCEC. Next, we investigated

whether Stxs affect the growth and survival of HRCEC. When
HRCEC were incubated with Stx1, their growth as assessed
by MTT assay was markedly reduced in a dose-dependent
manner (figure 3A), showing HRCEC to be very sensitive to the
cytotoxic effect of Stx1. The reduced cell growth in response to
treatment with Stx1 was due to cell death, as confirmed by
trypan blue staining (data not shown). Similar results were
obtained when HRCEC were incubated with Stx2 (figure 3B),
indicating that HRCEC are susceptible to both Stx1 and Stx2.
There was no significant difference in the effects of Stx1 and
Stx2 on HRCEC. Another population of HRCEC, derived from
a different volunteer (age 19 years), was similarly examined,
and identical results were obtained (data not shown).

Downloaded from http://jid.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015

Figure 2. Detection of Stx binding to HRCEC. A, HRCEC were
grown on chamber slides. After fixation with 100% cold acetone,
cells were incubated with 100 ng/mL Stx1 for 1 h at 47C. After
intensive washing, Stx1 bound to HRCEC was immunohistochemi-
cally stained with anti-Stx1 monoclonal antibody 13C4 (right). As
control, isotype-matched control immunoglobulin was also used (left).
B, HRCEC in suspension were incubated with (right) and without
(left) 100 ng/mL Stx2 for 1 h at 47C. After intensive washing, cells
were stained with anti-Stx2 monoclonal antibody 11E10 and analyzed
by flow cytometry. Histogram obtained was superimposed on that of
negative control (CNT; cells stained with isotype-matched control
immunoglobulin).

tribution of these molecules in normal renal tissue [29, 33, 34],

HRCEC in primary culture were thought to correspond to the
epithelia of distal tubules.
Stx binding to HRCEC. Since HRCEC were found to ex-
press CD77 on their cell surfaces, we investigated whether Stxs
bind to these cells. As shown in figure 2A, anti-Stx1 mono-
clonal antibody labeling of HRCEC pretreated with Stx1 was
demonstrated immunohistochemically. HRCEC not treated
with Stx1, on the other hand, failed to react with anti-Stx1
antibody (data not shown). Since the specificity of this antibody
was confirmed by immunostaining and ELISA (data not
shown), the positive staining of Stx1-pretreated HRCEC with
anti-Stx1 antibody was considered to reflect specific binding
of Stx1 to HRCEC.
The specific binding of Stx to HRCEC was also confirmed
by flow cytometry. As shown in figure 2B, antibody against Figure 3. Cytotoxic effects of Stx1 (A) and Stx2 (B) on HRCEC.
HRCEC (104 cells/100 mL of complete medium/well) were incubated
Stx2 clearly stained HRCEC pretreated with Stx2, whereas no with different concentrations of Stx1 or Stx2. After incubation, viable
staining was obtained when the antibody was reacted with cell numbers were estimated by MTT assay and are presented as ratio
untreated HRCEC. against non – Stx-treated cells.

/ 9d4a$$jy16 05-27-98 00:20:51 jinfa UC: J Infect

JID 1998;178 (July) Shiga Toxins Induce Apoptosis in Renal Tubules 181

Figure 4. Detection of DNA frag-

mentation in HRCEC treated with
Stx. A, HRCEC at 50% confluence
were cultured with (lanes 3 and 4)
or without (lane 2) 100 pg/mL Stx1.
After incubation, DNAs were ex-
tracted, and 1.5 mg of DNA from
each sample was electrophoresed in
1% agarose gel. As molecular
weight control, mixture of HindIII-
digested l DNA and HaeIII-digested

Downloaded from http://jid.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015

f DNA was also loaded on same
gel (lane 1). B, HRCEC treated with
(lower) and without (upper) 100 pg/
mL Stx2 were tested by TUNEL
method. Typical apoptotic cells
(dark color) are indicated by arrows.
C, After treatment with Stx1 (left) or
Stx2 (right) or without Stxs (center),
HRCEC were stained with propid-
ium iodide, and DNA contents were
examined. Each experiment was
done in triplicate, and averages of
ratio of subploid cells are shown.

To assess whether the cytotoxic effect described above is
specifically mediated by Stx, we examined the effects of anti-
bodies neutralizing Stxs. When 13C4 antibody, which neutral-
izes Stx1, was added to the culture, the cytotoxic effect of
Stx1 against HRCEC was markedly reduced (data not shown).
Similar results were obtained with a combination of Stx2 and
the anti-Stx2 monoclonal antibody 11F11 on HRCEC (data not
shown), thereby establishing that the cytotoxic effects were
directly mediated by Stxs.
Detection of apoptosis in the Stx-induced cell death of
HRCEC. As we have previously observed that Stxs induce
apoptosis on ACHN cells derived from human renal tubular
epithelial cancer cells [18], we examined the possibility that the
cytotoxic effect of Stxs on HRCEC involves an apoptotic path-
way. First, we tested whether cleavage of nuclear DNA occurs
during the Stx-induced death of HRCEC. As shown in figure
4A, DNA prepared from HRCEC pretreated with Stx1 exhibited
DNA ladder formation on agarose gel electrophoresis.
Cleavage of the nuclear DNA in HRCEC treated with Stx
was also confirmed by in situ detection by the TUNEL method,
as shown in figure 4B. After being treated with Stx2, a portion
of HRCEC clearly incorporated biotinylated nucleotides, indi-
cating the occurrence of DNA fragmentation induced by Stx2.
The TUNEL assay – positive cells were already detected at an
earlier time point when HRCEC were still trypan blue – nega-
tive (data not shown).
The cleavage of nuclear DNA in HRCEC treated with Stxs
was further confirmed by the detection of subploid cells with
propidium iodide staining (figure 4C). Again, no significant
difference was observed between the effect of Stx1 and that
of Stx2 on HRCEC in this experiment.
A 38-kDa protein, 7A6, detected by APO2.7 antibody was
recently identified as an early-stage marker of the apoptotic
process in various cells [28]. APO2.7 stained only a fraction
of untreated HRCEC, but the proportion of APO2.7-positive
HRCEC increased after treatment with Stx2 in a time-depen-
dent manner (figure 5). Equivalent results were obtained with
Stx1 treatment (data not shown). These findings demonstrate
clearly that apoptosis had indeed occurred in HRCEC following
treatment with Stxs.
Discussion
Although the capillary microangiopathy and endothelial
damage mediated by Stxs are generally believed to be the major

/ 9d4a$$jy16 05-27-98 00:20:51 jinfa UC: J Infect

182 Kiyokawa et al.
events triggering Stx-mediated HUS [3 – 8], several lines of
evidence have been reported that imply that renal tubular im-
pairment also contributes to the pathogenesis of HUS [12 – 18].
More recently, we have made several observations support-
ing this idea. First, using dual-color immunohistochemistry,
we have demonstrated that only tubular epithelial cells ex-
pressing CD24, a surface marker of renal epithelium repre-
senting distal tubules, can bind to Stxs (Mori T, et al., unpub-
lished data). Second, we had the opportunity to examine the
frozen renal autopsy tissue from a child who died of HUS
after STEC infection and found clear deposition of Stxs on a
significant portion of distal tubules as well as on endothelial
cells (Uchida H, et al., unpublished data). On the basis of
these findings, we hypothesized that renal tubular epithelium
might be another primary target of Stxs in vivo and should
be viewed as a major site of the renal dysfunction that occurs
in the early phase of HUS.
In fact, the data presented herein show clearly that HRCEC
are highly susceptible to the cytotoxic effect of Stxs. Based on
the distribution of various molecules in normal renal tissue [29,
33], HRCEC are assumed to correspond to renal epithelia of
the distal tubules. Collectively, our data indicate that distal
tubular epithelia, expressing CD24 and Gb3/CD77, are highly
susceptible to Stxs and can be directly damaged.
Stx consists of A and B subunits [35]. The B subunit works
as a receptor-binding site and facilitates the entry of the holo-
toxin into susceptible cells [36]. It is generally accepted that
the cytotoxic effect of Stxs is mediated by the A subunit, which
has RNA N-glycohydrolase activity and cleaves a specific ade-
nine residue on the 28S ribosomal subunit, resulting in inhibi-
tion of protein synthesis [36 – 39]. However, it has been re-
ported that Stxs induce apoptosis in various cells, such as
/ 9d4a$$jy16 05-27-98 00:20:51 jinfa
JID 1998;178 (July)
Figure 5. Induction of apoptotic antigen
7A6. After incubation with 100 pg/mL Stx2,
HRCEC were fixed and stained with either
APO2.7 monoclonal antibody (lower panels)
or isotype matched control immunoglobulin
(upper panels), then analyzed by flow cytome-
try. To identify positive cells more clearly,
fluorescence intensity was displayed as 2-di-
mensional histogram against side light scatter.
x axis, fluorescence intensity; y axis, side light
scatter.
Downloaded from http://jid.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
intestinal epithelium of rabbit, ACHN renal adenocarcinoma,
Burkitt’s lymphoma, Vero, and MDCK cells [18, 31, 40 – 44].
Consistent with these observations, we have demonstrated
herein that Stx also mediate apoptosis in normal renal cells in
primary culture. Fragmentation of genomic DNA of Stx-treated
HRCEC clearly indicates the presence of nucleosomal DNA
breakage, a phenomenon typical of apoptosis. In addition, the
expression of an apoptosis marker, the 7A6 molecule [28],
provides further evidence that apoptosis is indeed involved in
the mechanism of Stx-mediated cell death of normal human
tubular epithelial cells.
Under our experimental condition, Stxs caused apoptosis in
a portion of HRCEC. However, considering the several findings
that suggest the synergism between cytokines and Stxs in the
induction of apoptosis [6, 18, 45, 46], the apoptosis-inducing
effect of Stxs could be enhanced in vivo during STEC infection,
in which elevated plasma and urine concentrations of cytokines
have been observed [47 – 49]. In addition, previous reports de-
scribed a possible involvement of apoptosis in the process of
Stx-induced damage of other renal cells, such as glomerular
endothelia [4, 6, 42 – 45, 50]. These observations, together with
the results described herein, further emphasize the likelihood
of a pathologic role of Stx-mediated apoptosis of various renal
cells in HUS.
The difference in sensitivity to Stx-mediated apoptosis ob-
served among HRCEC could be due to heterogeneity in the
cell population. The factors or mechanisms determining the
sensitivity of HRCEC to apoptosis mediated by Stx remain to
be elucidated.
Although it has been clarified that Stxs are capable of inducing
apoptosis of various cell types from several animal species,
nothing is known about the mechanism of apoptotic signal trans-
UC: J Infect
JID 1998;178 (July) Shiga Toxins Induce Apoptosis in Renal Tubules
duction. Gb3/CD77 is known to serve as a functional receptor
for Stxs [19– 21], but its natural ligand and physiologic function
are also unknown. In the case of Burkitt’s lymphoma cells, since
the B subunit alone was found to be sufficient to induce
apoptosis, the presence of a signaling pathway mediating
apoptosis downstream from Gb3/CD77, which is independent
of the function of the A subunit, has been postulated [31, 51].
On the other hand, in the case of Vero cells, the B subunit
itself does not participate in cell killing, suggesting the involve-
ment of the A subunit in apoptosis induction [44]. That several
other members of the ribosome-inactivating protein family also
cause apoptosis in epithelial cells suggests the requirement of
protein synthesis inhibition for the apoptosis caused by these
agents [42, 44]. This apparent inconsistency may indicate the
presence of multiple mechanisms in Stx-mediated apoptosis.
The mechanism transducing the apoptotic signal mediated by
Stx binding to Gb3/CD77 on HRCEC will be investigated in
a future study.
In conclusion, the present as well as our past studies indicate
the involvement of renal tubular epithelial cell damage caused
by Stxs in the development of HUS. Although additional basic
studies must be done, the possibility of Stx-mediated apoptosis
in renal tubular epithelium, as described above, should provide
new approaches to understanding the pathogenesis of HUS and
to its prevention.
Acknowledgments
We thank R. Kannagi for the gift of CD77 monoclonal antibody
1A4 and M. Sone for excellent secretarial work.
References
1. Karmali MA, Petric M, Lim C, Fleming DC, Arbus GS, Lior H. The
association between idiopathic hemolytic uremic syndrome and infec-
tion by verotoxin producing Escherichia coli. J Infect Dis 1985; 151:
775 – 82.
2. Fong JS, De Chadarevian JP, Kaplan BS. Hemolytic uremic syndrome.
Current concepts and management. Pediatr Clin North Am 1982; 29:
835 – 56.
3. Richardson SE, Karmali MA, Becker LE, Smith CR. The histopathology
of the hemolytic uremic syndrome associated with verocytotoxin-pro-
ducing Escherichia coli infections. Hum Pathol 1988; 19:1102 – 8.
4. Obrig TG, Del-Vecchio PJ, Brown JE, et al. Direct cytotoxic action of
Shiga toxin on human vascular endothelial cells. Infect Immun 1988;
56:2373 – 8.
5. Tesh VL, Samuel JE, Perera LP, Sharefkin JB, O’Brien AD. Evaluation
of the role of Shiga and Shiga-like toxins in mediating direct damage
to human vascular endothelial cells. J Infect Dis 1991; 164:344 – 52.
6. Kaye SA, Louise CB, Boyd B, Lingwood CA, Obrig TG. Shiga toxin –
associated hemolytic uremic syndrome: interleukin-1b enhancement of
Shiga toxin cytotoxicity toward human vascular endothelial cells in
vitro. Infect Immun 1993; 61:3886 – 91.
7. Obrig TG, Louise CB, Lingwood CA, Boyd B, Barley-Maloney L, Daniel
TO. Endothelial heterogeneity in Shiga toxin receptors and responses.
J Biol Chem 1993; 268:15484 – 8.
/ 9d4a$$jy16 05-27-98 00:20:51 jinfa
183
8. Kaplan BS, Cleary TG, Obrig TG. Recent advances in understanding the
pathogenesis of the hemolytic uremic syndromes. Pediatr Nephrol 1990;
4:276 – 83.
9. Arends MJ, Harrison DJ. Novel histopathologic findings in a surviving
case of hemolytic uremic syndrome after bone marrow transplantation.
Hum Pathol 1989; 20:89 – 91.
10. Laurence J, Mitra D, Steiner M, Staiano-Coico L, Jaffe E. Plasma from
patients with idiopathic and human immunodeficiency virus – associated
thrombotic thrombocytopenic purpura induces apoptosis in microvascu-
lar endothelial cells. Blood 1996; 87:3245 – 54.
11. Mitra D, Jaffe EA, Weksler B, Hajjar KA, Soderland C, Laurence J.
Thrombotic thrombocytopenic purpura and sporadic hemolytic-uremic
syndrome plasmas induce apoptosis in restricted lineages of human
microvascular endothelial cells. Blood 1997; 89:1224 – 34.
12. Wadolkowski EA, Sung LM, Burris JA, Samuel JE, O’Brien AD. Acute
renal tubular necrosis and death of mice orally infected with Escherichia
coli strains that produce Shiga-like toxin type II. Infect Immun 1990;
Downloaded from http://jid.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
58:3959 – 65.
13. Lindgren SW, Melton AR, O’Brien AD. Virulence of enterohemorrhagic
Escherichia coli O91:H21 clinical isolates in an orally infected mouse
model. Infect Immun 1993; 61:3832 – 42.
14. Tesh VL, Burris JA, Owens JW, et al. Comparison of the relative toxicities
of Shiga-like toxins type I and type II for mice. Infect Immun 1993;
61:3392 – 402.
15. Oosterwijk E, Kalisiak A, Wakka JC, Scheinberg DA, Old LJ. Monoclonal
antibodies against Gala1-4Galb1-4Glc(Pk, CD77) produced with a syn-
thetic glycoconjugate as immunogen: reactivity with carbohydrates,
with fresh frozen human tissues and hematopoietic tumors. Int J Cancer
1991; 48:848 – 54.
16. Lingwood CA: Verotoxin-binding in human renal sections. Nephron 1994;
66:21 – 8.
17. Takeda T, Dohi S, Igarashi T, Yamanaka T, Yoshiya K, Kobayashi N.
Impairment by verotoxin of tubular function contributes to the renal
damage seen in haemolytic uremic syndrome. J Infect 1993; 27:339 –
41.
18. Taguchi T, Uchida H, Kiyokawa N, et al. Verotoxins induce apoptosis in
human renal tubular epithelium derived cells. Kidney Int 1998; 53(6).
19. Jacewicz M, Clausen H, Nudelman E, Donohue-Rolfe A, Keusch GT.
Pathogenesis of Shigella diarrhea. XI. Isolation of a Shigella toxin –
binding glycolipid from rabbit jejunum and HeLa cells and its identifi-
cation as globotriaosylceramide. J Exp Med 1986; 163:1391 – 404.
20. Lindberg AA, Brown JE, Stromberg N, Westling-Ryd M, Schultz JE,
Karlsson KA. Identification of the carbohydrate receptor for Shiga toxin
produced by Shigella dysenteriae type 1. J Biol Chem 1987; 262:1779 –
85.
21. Lingwood CA, Law H, Richardson S, et al. Glycolipid binding of purified
and recombinant Escherichia coli produced verotoxin in vitro. J Biol
Chem 1987; 262:8834 – 9.
22. Noda M, Yutsudo T, Nakabayashi N, Hirayama T, Takeda Y. Purification
and some properties of Shiga-like toxin from Escherichia coli O157:
H7 that is immunologically identical to Shiga toxin. Microb Pathog
1987; 2:339 – 49.
23. Oku Y, Yutsudo T, Hirayama T, O’Brien AD, Takeda Y. Purification and
some properties of a Vero toxin from a human strain of Escherichia
coli that is immunologically related to Shiga-like toxin II (VT2). Microb
Pathog 1989; 6:113 – 22.
24. Fujimoto J, Ishimoto K, Kiyokawa N, Tanaka S, Ishii E, Hata J. Immuno-
cytological and immunochemical analysis on the common acute lym-
phoblastic leukemia antigen (CALLA): evidence that CALLA on ALL
cells and granulocytes are structurally related. Hybridoma 1988; 7:
227 – 36.
25. Kokai Y, Ishii Y, Kikuchi K. Characterization of two distinct antigens
expressed on either resting or activated human B cells as defined by
monoclonal antibodies. Clin Exp Immunol 1986; 64:382 – 91.
UC: J Infect
184 Kiyokawa et al.
26. Strockbine NA, Marques LR, Holmes RK, O’Brien AD. Characterization
of monoclonal antibodies against Shiga-like toxin from Escherichia
coli. Infect Immun 1985; 50:695 – 700.
27. Perera LP, Marques LR, O’Brien AD. Isolation and characterization of
monoclonal antibodies to Shiga-like toxin II of enterohemorrhagic Esch-
erichia coli and use of the monoclonal antibodies in a colony enzyme-
linked immunosorbent assay. J Clin Microbiol 1988; 26:2127 – 31.
28. Zhang C, Ao Z, Seth A, Schlossman SF. A mitochondrial membrane
protein defined by a novel monoclonal antibody is preferentially de-
tected in apoptotic cells. J Immunol 1996; 157:3980 – 7.
29. Ishii E, Fujimoto J, Tanaka S, Hata J. Immunohistochemical analysis on
normal nephrogenesis and Wilms’ tumor using monoclonal antibodies
reactive with lymphohaemopoietic antigens. Virchows Arch A 1987;
411:315 – 22.
30. Hansen MB, Nielsen SE, Berg K. Re-examination and further development
of a precise and rapid dye method for measuring cell growth/cell kill.
J Immunol Methods 1989; 119:203 – 10.
31. Mangeney M, Lingwood CA, Taga S, Caillou B, Tursz T, Wiels J.
Apoptosis induced in Burkitt’s lymphoma cells via Gb3/CD77, a glyco-
lipid antigen. Cancer Res 1993; 53:5314 – 9.
32. Gong J, Traganos F, Darzynkiewicz Z. A selective procedure for DNA
extraction from apoptotic cells applicable for gel electrophoresis and
flow cytometry. Anal Biochem 1994; 218:314 – 9.
33. Platt JL, LeBien TW, Michael AF. Stages of renal ontogenesis identified by
monoclonal antibodies reactive with lymphohemopoietic differentiation
antigens. J Exp Med 1983; 157:155 – 72.
34. Fina L, Molgaard HV, Robertson D, et al. Expression of the CD34 gene
in vascular endothelial cells. Blood 1990; 75:2417 – 26.
35. Lingwood CA. Verotoxins and their glycolipid receptors. Adv Lipid Res
1993; 25:189 – 212.
36. Donohue-Rolfe A, Keusch GT, Edson C, Thorley-Lawson D, Jacewicz
M. Pathogenesis of Shigella diarrhea. IX. Simplified high yield purifica-
tion of Shigella toxin and characterization of subunit composition and
function by the use of subunit-specific monoclonal and polyclonal anti-
bodies. J Exp Med 1984; 160:1767 – 81.
37. Obrig TG, Moran TP, Brown JE. The mode of action of Shiga toxin on
peptide elongation of eukaryotic protein synthesis. Biochem J 1987;
244:287 – 94.
38. Endo Y, Tsurugi K, Yutsudo T, Takeda Y, Ogasawara T, Igarashi K. Site
of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of
/ 9d4a$$jy16 05-27-98 00:20:51 jinfa
JID 1998;178 (July)
Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of
the toxins. Eur J Biochem 1988; 171:45 – 50.
39. Saxena SK, O’Brien AD, Ackerman EJ. Shiga toxin, Shiga-like toxin II
variant, and ricin are all single-site RNA N-glycosidases of 28 S RNA
when microinjected into Xenopus oocytes. J Biol Chem 1989; 264:596 –
601.
40. Keenan KP, Sharpnack DD, Collins H, Formal SB, O’Brien AD. Morpho-
logic evaluation of the effects of Shiga toxin and E. coli Shiga-like
toxin on the rabbit intestine. Am J Pathol 1986; 125:69 – 80.
41. Pai CH, Kelly JK, Meyers GL. Experimental infection of infant rabbits with
verotoxin-producing Escherichia coli. Infect Immun 1986;51:16–23.
42. Sandvig K, van Deurs B. Toxin-induced cell lysis: protection by 3-methyl-
adenine and cycloheximide. Exp Cell Res 1992; 200:253 – 62.
43. Inward CD, Williams J, Chant I, et al. Verocytotoxin-1 induces apoptosis
in vero cells. J Infect 1995; 30:213 – 8.
44. Williams JM, Lea N, Lord JM, Roberts LM, Milford DV, Taylor CM.
Comparison of ribosome-inactivating proteins in the induction of
Downloaded from http://jid.oxfordjournals.org/ at UNIVERSITY OF ARIZONA on May 30, 2015
apoptosis. Toxicol Lett 1997; 91:121 – 7.
45. Polunovsky VA, Wendt CH, Ingbar DH, Peterson MS, Bitterman PB.
Induction of endothelial cell apoptosis by TNF alpha: modulation by
inhibitors of protein synthesis. Exp Cell Res 1994; 214:584 – 94.
46. Eissner G, Kohlhuber F, Grell M, et al. Critical involvement of transmem-
brane tumor necrosis factor-alpha in endothelial programmed cell death
mediated by ionizing radiation and bacterial endotoxin. Blood 1995; 86:
4184 – 93.
47. Fitzpatrick MM, Shah V, Trompeter RS, Dillon MJ, Barratt TM. Interleu-
kin-8 and polymorphoneutrophil leucocyte activation in hemolytic ure-
mic syndrome of childhood. Kidney Int 1992; 42:951 – 6.
48. van de Kar NC, Sauerwein RW, Demacker PN, Grau GE, van Hinsbergh
VW, Monnens LA. Plasma cytokine levels in hemolytic uremic syn-
drome. Nephron 1995; 71:309 – 13.
49. Karpman D, Andreasson A, Thysell H, Kaplan BS, Svanborg C. Cytokines
in childhood hemolytic uremic syndrome and thrombotic thrombocyto-
penic purpura. Pediatr Nephrol 1995; 9:694 – 9.
50. Mahan JD, McAllister C, Karmali M. Verocytotoxin-1 (VT-1) induction
of apoptosis in human glomerular capillary endothelial cells (GCEC) in
vitro is dependent on cytokines, cell confluence, and cell cycle [abstract
A2067]. J Am Soc Nephrol 1996; 7:1661.
51. Taga S, Carlier K, Mishal Z, et al. Intracellular signaling events in
CD77-mediated apoptosis of Burkitt’s lymphoma cells. Blood 1997;
90:2757 – 67.
UC: J Infect