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The effects of phytohemagglutinin on the growth and histochemical properties of mammalian cells in tissue culture.

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The Effects of Phytohemagglutinin on the Growth
and Histochemical Properties of Mammalian
Cells in Tissue Culture
' j 2
Department of Histology, Temple University School of Dentistry,
Philadelphia, Pennsylvania
Phytohemagglutinin-P (PHA-P) exhibited inhibitory action on the
growth of two human epithelioid cell lines, HeLa and L-132, and on the G929 strain
of mouse fibroblasts. Growth of stationary cultures was measured spectrophotometrically by DNA production after 96 hours incubation. The mitotic indices of the three
cell lines were reduced significantly by PHA-P after 72 hours incubation.
Histochemical studies demonstrate depressed production of cytoplasmic RNA in
72-hour HeLa cultures after treatment with PHA-P, with the appearance of prominent,
PAS-positive, diastase-fast granules in the cytoplasm. Bound lipid aggregates increased
i n amount in the cytoplasm of HeLa cells after PHA-P treatment. The PAS-positive,
diastase-fast granules were also found in the cytoplasm of the L-132 and L-929 cell
lines after treatment, but cytoplasmic RNA remained unchanged qualitatively. Alkaline phosphatase was slight in amount in HeLa cells and was reduced to trace amounts
after PHA-P treatment. L-132 cells showed moderate to heavy concentration of alkaline phosphatase, which remained unchanged qualitatively after PHA-P treatment.
The enzyme could not be demonstrated in the L-929 fibroblasts.
Phytohemagglutinin (PHA) is an extract (Rigas and Osgood, ' 5 5 ) of the red
kidney bean (Phaseolus vulgaris) which
agglutinates erythrocytes in low concentrations and leukocytes in high concentrations. Nowell ('60) observed that PHA
causes mitotic activity in the leukocytes of
peripheral blood in tissue culture. Mitoses
in leukocyte cultures are known to be preceded by the enlargement of small lymphocytes and their transformation to blastoid
cells (Carstairs, '62; MacKinney, Stohlman
and Brecher, '62). While the mitotic index
of normal leukocyte cultures is high after
PHA treatment, not all the lymphocytes of
an affected culture undergo blastoid cell
formation and mitosis (Cooper, Barkhan
and Hale, '63), and in certain conditions,
such as chronic lymphocytic leukemia
(Bernard, Geraldes and Boiron, '64), relatively few or no blastoid cells develop. The
stimulatory effects of PHA on leukocytes
have been reviewed by Robbins ('64).
There is, in addition, evidence for in vivo
stimulation of lymphatic tissue by PHA,
although other findings indicate suppression of the primary and secondary immune
responses in rodents, and inhibition of
phagocytosis in the reticuloendothelial sysANAT.REC.,162: 459-466,
tem of mice (Lozzio, '67; Jennings and
Oates, '67). PHA has been reported to
cause increased mitoses in the basal cell
layer of human skin explants (Sarkamy
and Caron, '65), and acanthosis in guinea
pig skin after intradennal injection (Sarkamy and Caron, '66). The findings of
Ioachim ('66) indicate that PHA causes
clumping of L-929 fibroblasts and other
mouse, rat and human cell lines and
changes in the morphology of these cell
lines in tissue culture. However, the treated
cultures were found to be viable and their
mitotic indices exceeded those of the controls.
In the present study PHA was added to
cultures of three established cell-lines :
HeLa, derived from a human epidermoid
carcinoma of the cervix; L-132, derived
from human embryonic lung; and the
L-929 strain of mouse fibroblasts. In contrast to the growth-stimulating action seen
on lymphocytes, PHA inhibited the growth
of these cell cultures and depressed their
mitotic indices.
1 Supported by General Research
2This paper was presented in part
Session of the American Association
at New Orleans, Louisiana, April 10,
Support grant
at the Annual
of Anatomists
D N A cell cultures. All cultures were
grown in Blake flasks as stationary monolayer cultures. HeLa and L-929 were cultured in Eagle's minimum essential medium with 15% horse serum and 0.03%
glutamine. L-132 cells were grown in LY
medium (Difco) with the same concentrations of horse serum and glutamine. Each
experiment consisted of a set of PHAtreated cultures and control cultures. The
former contained an added amount of
phytohemagglutinin-P (PHA-P) (Wilson,
'66) in a concentration of either 0.02 ml/
ml, 0.01 ml/ml or 0.005 ml/ml. The controls consisted either of untreated cultures,
cultures containing an equivalent amount
of triple-distilled water, or cultures containing an equivalent amount of PHA-P
suspension resulting from denaturation by
heating at 100°C for 15 minutes, or of
the clear supernatant from the denatured
suspension. Each flask was inoculated with
10' cells/lO ml culture medium, incubated
at 36°C for four days, after which the medium was removed from each culture flask
and centrifuged. Cells adhering to the
glass of HeLa and L-132 culture flasks
were lightly trypsinized (0.1 ml 10%
trypsin in 5 mlO.85% NaCl), followed by
one or two washings of 3 to 5 ml normal
saline. L-929 cultures were treated similarly but instead of trypsinization the cells
were scraped free from the glass with a
rubber policeman. Cells which had been
removed from the original medium by centrifuging were added to those recovered by
trypsinization or scraping. PHA tended to
agglutinate the cells (especially HeLa),
which then floated free from the glass.
Growth of cell cultures was estimated
as a function of total DNA production
(Salzman, '59) by the Paul ('56) modification of the micro-method of Ceriotti, using
hot perchloric acid extraction and indol
color formation. Optical density was read
in the Perkin-Elmer spectrophotometer at
489 mu. The O.D. value at 520 mu was
subtracted from that at the wave length
specific for DNA in order to eliminate interference from substances with wave
lengths outside the DNA optical density
Histochemical procedures. All cultures
were inoculated with 10' cells/lO ml me-
dium and grown for 72 hours at 36°C. The
culture media were the same as those used
in the DNA growth experiments, and all
treated cultures contained PHA-P in concentrations stated for specific experiments.
(a) RNA. Trypsinized cells were washed
three times in Hanks balanced salt solution (BSS) and fixed five minutes in either
neutral buffered formalin or Carnoy's fixative, dehydrated and embedded in paraffin (30 to 40 minutes). Paraffi sections
were brought to water and incubated in
ribonuclease (Pearse, '61), 100 mg/100
ml demineralized water, at 36°C for two
hours, accompanied by control sections in
demineralized water. Sections were then
stained with 1 % toluidine blue.
(b) Alkaline phosphatase. The method
described by Gomori ('52) and modified
by Merchant, Kahn and Murphy ('60)
was adapted for use in cell suspensions.
The trypsinized cells, washed three times
in BSS, were fixed for five minutes in neutral buffered formalin. This was followed
by two centrifuge washings in demineralized water. Cell suspensions were reacted
in a solution of the substrate (sodium
alpha-naphthyl acid phosphate) and pchloro-o-toluidine diazonium salt, and in
control solution of diazonium salt alone,
for 30 minutes at room temperature, followed by two more centrifuge washings in
demineralized water. Cell suspensionswere
mounted on microscope slides in glycerogel.
(c) PAS reaction. The trypsinized cells
were washed three times in BSS and once
in normal saline, fixed five minutes in
Carnoy's fixative, dehydrated and embedded in paraffin (30 to 40 minutes). Sections were oxidized with 0.5% periodic
acid and stained with Schiffs reagent in
the usual PAS procedure. To determine if
glycogen was present, deparaffinized sections were coated with celloidin and incubated in 1% diastase in neutral buffered
saline for one hour at 37°C with suitable
control sections.
( d ) Bound lipid. In preparation for the
method of Berenbaum ('58) trypsinized
cells were washed three times in BSS,fixed
for five minutes in Carnoy's fixative, de3 Phytohemagglutinin-P (Difco). approx. 100 mg
reconstituted with 5 ml tripledistilled water.
4 Ribonuclease (Nutritional Biochemical
Corp. ),
crystalline, 5 x.
hydrated and embedded in paraffin. De- tures at 36°C in Leighton tubes till forparaffiized sections were then washed mation of a monolayer on the glass, then
overnight in running tap water, dehydrated drawing off the medium and adding, in
in absolute acetone and stained in Sudan 2 ml quantities, dilutions of PHA-P, in regBlack B in acetone at 37°C for periods of ular culture medium in concentrations
six hours and 24 hours. For the modifica- ranging from 0.0025 ml/ml to 0.02 ml/
tion of the method of Ackerman ('52) ml, followed by observation during the next
fresh cell suspensions were used. Whole, 72 hours of incubation. Slight morphologitrypsinized cells were washed three times cal changes, such as withdrawal of some
in BSS and fixed five minutes in neutral cytoplasmic processes, or rounding of some
buffered formalin. Hydrolysis in 25% cells, were seen at the 0.0025 ml concenacetic acid for five minutes followed, suc- tration, with increased rounding of cells
ceeded by two centrifuge washings in de- through the 0.02 ml concentration. Cell
mineralized water. Cell suspensions were disintegration increased at the higher conthen stained in Sudan Black B in 70% centrations, but cytotoxicity did not apethanol, followed by four centrifuge wash- pear to reach the plus two grades described
ings in 70% ethanol. Cells were mounted by Syverton or by Toplin ('59). HeLa apon microscope slides in glycerogel.
peared to be the most resistant to cyto( e ) Baker's pyridine extraction. PHA- toxicity and L-132 the most susceptible.
P-treated cultures and control cultures
Cell viability was determined by withwere trypsinized, washed three times in drawing the PHA-medium after 72 hours,
BSS, centrifuged and fked 20 hours in and adding to the tubes a 1:5000 solution
diluted Bouin's fixative according to the of neutral red in sterile Earle's solution.
procedure outlined by Pearse ('61 ). Treat- Most cells adhering to the glass were capaed and control cells were extracted 24 ble of showing supravital staining with the
hours in pyridine at 60°C. Both treated dye. The range of viability was within the
and control cells were washed in tap water, limits, normal for control cultures, 85dehydrated and embedded in paraffin (30- 95% viable cells, for all dilutions of PHA40 minutes) and stained by the PAS pro- P tested.
After the 72-hour treatment of the cells
( f ) Hyaluponidase reaction. Paraffin sec- with the various dilutions of PHA-P, the
tions of PHA-P-treated cultures were pre- ability of the cultures to recover normal
pared as described for the PAS reaction morphology and growth was tested by rebut were deparaffinized and incubated with moving the PHA-medium, washing the cultesticular hyaluronidase (approximately ture three times with BSS and followed by
100 USP U./ml) in 0.1 M phosphate buf- addition of fresh medium. All cultures
fer (pH 7) for 24 hours at 37"C, according regrew normal cells during the first 24
to the method of Gersh and Catchpole hours.
('49) for depolymerization of glycoprotein
Mitotic index. The mitotic index of
ground substance. Washed sections were each cell-line was determined by counting
then stained by the PAS procedure.
1000 cells, in sections embedded in paraf( g ) Other staining reactions. These in- fin and stained with iron hematoxylin, to
cluded the Baker modification of the Mil- determine the number of mitotic figures.
Ion reaction (Barka and Anderson, ' 6 3 ) , Normal control cultures of L132, L-929
the Nile blue stain of Lillie ('56), iron and HeLa were found to have mitotic inhematoxylin and acid Alcian blue (1% dices of 2.67, 1.7 and 0.96 respectively,
in 0.1 N HC1, pH 2). Trypsinized cells and these were reduced in the drug-treated
were washed three times in BSS, fixed five cultures, over the range of PHA concentraminutes in Carnoy's fixative, dehydrated tions used above, to 0.87, 0.30 and 0.16
and embedded in paraffin (30-40 minutes). respectively, or one-third to one-sixth the
control values.
D N A growth measurements. It can be
Cytotoxicity and cell viability. Prelim- seen in table 1 that PHA-P, 0.02 ml/ml,
inary tests for cytotoxicity and viability
hyaluronidase (Worthington Biochemwere carried out by incubating the cell cul- ical5 Testicular
Optical densities a t 489 mp f o r D N A content of
HeLa cell cultures (loa cells/lO m l ) a f t e r
addition of phytohemagglutinin-P.
Incubation at 36" C f o r 96 hours
0.232 1
0.121 2
< 0.05
< 0.05
< 0.001
< 0.025
> 0.05
< 0.005
< 0.005
< 0.05
< 0.005
< 0.001
lMean value of three cultures.
2 Mean value of two cultures.
SMean value of four cultures.
4 Equivalent amount of triple-distilled water added
to control culture.
5 Equivalent amount of supernatant from PHA-P
(denatured at 100°C for 15 minutes) added to control
significantly reduced the growth of HeLa
cells. This inhibition was the same whether
controls were untreated, or treated by the
addition of triple-distilled water or the supernatant from the heat-denatured drug.
For lower concentrations of PHA-P (0.01
ml/ml and 0.005 ml/ml) inhibition of
HeLa cell growth was suggested but not
significant according to the probability
value (P) of the Student t test. The L-132
cells proved to be more sensitive to PHA-P,
and table 2 shows consistent inhibition at
the 0.005 ml/ml and 0.01 ml/ml concentrations. Here it is seen also that the heat-
denatured suspension of PHA-P could be
used effectively as a control in two experiments. The L-929 fibroblasts exhibited inhibition of growth at the 0.02 ml/ml,
0.01 ml/ml and 0.005 ml/ml concentrations of PHA-P, as shown in table 3. This
was consistently significant, except in experiments 9 and 10, in which the controls
showed much variability in DNA synthesis.
The whole suspension of the heat-denatured drug was used in the controls in
these experiments, and perhaps the suspended particles in the culture medium
interfered with metabolism at the cell surfaces. When triple-distilled water or the
clear supernatant from the heat-denatured
PHA was employed in control cultures, the
experimental cultures showed consistently
significant inhibition of cell growth.
Histochemistry. Staining for RNA indicated that 72-hour HeLa cultures, treated
with PHA-P, 0.02 ml/ml, showed an even
distribution of RNA in the cytoplasm, but
intensity of staining was much heavier in
untreated control cultures. Qualitatively,
four separate experiments indicated a decreased amount of RNA synthesis after
PHA-P treatment. Nuclei of control HeLa
cells were also much more heavily stained
than those of the treated cells, while nucleoli stained the same in both. L-929 and
L-132 cell cultures showed heavy cytoplasmic staining for RNA, and this was true
for the 0.01 ml/ml and 0.02 ml/ml concentrations of PHA-P. Control cultures of
L-929 and G132 cells showed no qualitative difference in RNA staining.
Optical densities at 489 mp f o r D N A content o f human embryonic lung (L-132) cultures
(108 cells/lO m l ) a f t e r addition of phytohemagglutinin-P. Incubation at 36'C for 96 hours
0.395 1A
0.253 1,4
ml jml )
0.100 4
< 0.001
< 0.01
< 0.01
< 0.001
< 0.005
< 0.001
< 0.05
< 0.001
1 Equivalent amount of triple-distilled water added to control culture.
9Equivalent amount of PHA-P (denatured at 100°C for 15 minutes) added to control culture.
3 Mean value for three cultures.
4 Mean value for four cultures.
Optical densities at 489 mjt for D N A content of mouse fibroblast (L-929)cultures (lo* cells/
1 0 m l ) after addition o f phytohemagglutinin-P. Incubation at 36°C f o r 96 hours
0.178 s ~ l
0.1723 ~ 1
0.319 52
( O.D. 1
0.129 5
0.162 '
0.072 '
< 0.025
< 0.025
< 0.025
< 0.001
< 0.005
< 0.05
< 0.01
< 0.01
> 0.2
> 0.2
< 0.001
< 0.05
< 0.05
< 0.001
'Equivalent amount of PHA-P (denatured at 100°C for 15 minutes) added to control culture.
Equivalent amount of supernatant from PHA-P (denatured at 100°C for 15 minutes) added to
control culture.
3 Mean value of three cultures.
4 Mean value of two cultures.
5 Mean value of four cultures.
Alkaline phosphatase content of HeLa
cells qualitatively was diminished in cultures treated with PHA-P, 0.02 ml/ml. In
four separate experiments, alkaline phosphatase was distributed in the cytoplasm
of most control cells in slight to moderate
amounts, and in occasional groups of cells
in heavily stained areas, especially just inside the plasma membrane. PHA-P-treated
cells showed a similar distribution, but alkaline phosphatase was much more lightly
stained even in those areas where it had
concentrated more abundantly, and many
cells showed no staining or only trace
amounts. This was in contrast to the alkaline phosphatase content of C132 cells,
which consistently showed heavy cytoplasmic staining both in control cultures,
and in those treated with PHA-P at the
0.02, 0.01 and 0.005 ml/ml concentrations. L-929 cells did not stain for alkaline
phosphatase, either in control or treated
The PAS reaction for polysaccharides
demonstrated red cytoplasmic granules in
HeLa cells both in control cultures and
cultures treated with PHA-P, 0.02 ml/ml.
The granules in the treated cells appeared
to be coarser and more prominent. Hydrolysis in 1% diastase before PAS staining
removed all the red granules from the cy-
toplasm of control cells, while many granules persisted in the cells of cultures
containing PHA-P. This observation was
confirmed in four separate experiments.
Both L-132 and L-929 cells showed PASpositive granules in the cytoplasm of cells
treated with PHA-P at the 0.02, 0.01 and
0.005 ml/ml concentrations. These granules were finer and present in fewer cells
at the lower concentrations of PHA-P,
while at the 0.02 ml/ml concentration
they were larger and resembled those of
the treated HeLa cells. As in the HeLa
cultures, the PAS-positive granules of the
treated L-132 and L-929 cells also were
found to be diastase-fast.
Reaction of PHA-P-treated cell cultures
with hyaluronidase failed to remove the
PAS-positive granules from the cytoplasm.
This experiment was repeated with three
cultures of L-929 cells at the 0.02, 0.01
and 0.005 ml/ml concentrations of PHAP. The granules were clearly identifiable
in all cases, although the culture treated
with the 0.02 ml/ml concentration gave
somewhat fainter PAS staining than the
hyaluronidase control after enzymatic action. L-132 cells, treated with the 0.02 ml/
ml concentration of PHA-P, showed no
difference in PAS staining after the hyaluronidase reaction.
Pyridine extraction did not alter the
PAS staining of cytoplasmic granules of
L-929 cells treated with PHA-P, 0.02 ml/
ml. This experiment was repeated with the
L-132 cells also treated with the 0.02 ml/
ml drug concentration, with identical results.
Consistent with the negative findings
for hyaluronidase and pyridine extraction,
the Millon reaction repeatedly failed to
demonstrate these granules in the HeLa
cells, thus excluding protein, and Alcian
blue failed to demonstrate acid mucopolysaccharide granules in either treated or
control HeLa cells. Nile blue A failed to
demonstrate lipofuscin granules in the
same cells.
The hydration method of Berenbaum
('58) showed that the cytoplasm of HeLa
cells from control cultures and cultures
treated with PHA-P, 0.02 ml/rnl, contained fine, rather evenly distributed black
granules, indicating bound lipid in both
types of cultures in approximately equal
amounts. However, the method of Ackerman ('52), modified for use in cell suspensions, demonstrated aggregates of
large, black granules in the cytoplasm of
many of the cells treated with the 0.02
ml/ml concentration of PHA-P. Some of
the control cells had similar granules, but
these were comparatively few in number.
This would indicate that PHA may induce
increased amounts of bound lipid in HeLa
cells. Although bound lipid in the oxidized
state would be PAS-positive, the granules
did not correspond exactly to the PAS-positive granules described in the PHA-Ptreated cells.
The cytochemical changes in leukocytes
after PHA stimulation have been investigated rather extensively, and it is known
that alkaline phosphatase decreases in
amount in polymorphonucleocytes after
PHA treatment, and degeneration of these
cells proceeds more quickly. The "blast"
cells of PHA-stimulated cultures are substantially negative for alkaline phosphatase (Quaglino, Hayhoe and Flemens, '62).
After PHA treatment, RNA synthesis has
been found to begin within the first few
hours, and DNA synthesis in mononuclear
cells in 24-48 hours (Killander and Rig-
ler, '65). Uridine-5-3H labeling suggests
that RNA synthesis occurs first in the nucleus and later in the cytoplasm in stimulated leukocyte cultures (Winter and Yoffey, '65). There may be a difference in the
type of RNA molecule in lymphocytes
stimulated by PHA and bacterial antigen,
the former stimulating non-ribosomal RNA
and the latter stimulating a precursor to
ribosomal RNA (Cooper and Rubin, '65).
The transformation of lymphocytes and the
accompanying RNA and DNA changes by
PHA are similar to those described more
recently for another stimulator of lymphocyte metabolism, pokeweed mitogen, which
however produces two types of stimulated
lymphocytes, one of which possesses PASpositive, diastase-resistant granules. ( Chessin et al., '66; Schwarz, '68). In addition, it
should be noted that PHA, in high concentrations, depresses lymphocyte transformation and their uptake of labeled thymidine (Wilson, '66).
Glycogen has been found to accumulate
in transforming lymphocytes prior to DNA
synthesis (Hayhoe and Quaglino, '65). It
reaches a maximum in lymphocytes at 1824 hours and persists for 48 hours. Most
"blast" cells are negative, their energy for
transformation having been derived from
glycogen which was then depleted (Quaglino, Hayhoe and Flemens, '62).
Razavi ('66) found that a fluoresceinlabeled PHA moiety localized in the cytoplasm of lymphocyte "blast" cells and granulocytes. It is possible that the mammalian
cells showing PAS-positive granules in the
present study behave like the granulocytes
of PHA-stimulated cultures. The possibility
that these granules represent incompletely
metabolized PHA-P in the cytoplasm merits further investigation. The results of the
present study, using diastase, pyridine extraction and the hyaluronidase and Millon
reactions, would seem to exclude glycogen,
glycolipid, and glycoprotein from constituting the substance of the diastase-fast granules, although some glycogen granules
were present in the treated HeLa cells.
The agglutinating and mitogenic actions
of PHA can be differentiated by heating
(Holland and Holland, '65) or erythrocyte
absorption (Barkhan and Ballas, '63), and
possible separate factors for agglutination
and mitogenic action have been indicated
by electrophoresis (Marshall and Norins.
' 6 5 ) . PHA, absorbed by erythrocytes, exhibits agglutination of S-180 cells, an action distinct from erythrocyte agglutination
(Tunis, '64). Furthermore, unabsorbed
PHA-P has been shown to agglutinate many
cell types, including erythrocytes and leukocytes of various species, C3H sarcoma,
Ehrlich ascites tumor and L fibroblasts
(Steck and Wallach, '65). These cells have
one or more binding sites in common with
PHA. Changed electrophoretic mobilities of
lymphocytes treated with PHA suggest adsorption of the drug (Vassar and Culling,
'64). In view of these findings, the inhibitory action of PHA-P on HeLa, L-929 and
L-132 cells may be due to interference of
the agglutinating factor of PHA at the
plasma membrane.
Stimulation of lymphocyte '%last" cell
formation by PHA is similar to stimulation
in vitro of previously sensitized lymphocytes by antigen (Benezra, Gery and Davies, '67) or to stimulation of lymphocytes
in the homograft reaction (Gowans, McGregor, Cowen and Ford, '62), but the difference in the RNA molecule produced by
antigen and PHA indicates a different
mechanism of action for each (Cooper
and Rubin, '65). However, the mitogenic
action of PHA is neutralized by antiserum
to PHA-M, the substance itself being antigenic (Byrd, Finley, Finley and McClure,
'64), and human splenic extract inhibits
or suppresses the transformation of leukocytes by PHA (Pegrum, '65). The stimulating effects of anti-leukocyte antiserum on
leukocytes resemble the action of PHA in
potency and time sequence (Grasbeck,
Nordman and de la Chapelle, '64). In the
present study, PHA possibly may be acting
like an antibody (agglutinin) by coating
antigenic sites on the plasma membrane
and thereby interfering with cellular metabolism.
The resemblance of PHA stimulation of
lymphocytes to antigenic stimulation, and
the ability of PHA to agglutinate cells other
than blood cells by a separate agglutinating
factor, suggest a dual action which should
be investigated further. If the agglutinating factor is indeed responsible for the
inhibition of cell growth in tissue culture,
the possibility of its selective inhibitory
action on certain cells, while lymphatic
cells are either stimulated or relatively unaffected, suggests potential therapeutic
The author expresses his appreciation
and thanks to Mrs. Nevart Gulezian and
Miss Helen Ruane for their technical assistance.
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effect, properties, mammalia, growth, phytohemagglutinin, culture, tissue, cells, histochemical
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